Methods of nucleic acid amplification and sequencing

ABSTRACT

Methods for amplification and sequencing of at least one nucleic acid comprising the following steps: (1) forming at least one nucleic acid template comprising the nucleic acid(s) to be amplified or sequenced, wherein said nucleic acid(s) contains at the 5′ end an oligonucleotide sequence Y and at the 3′ end an oligonucleotide sequence Z and, in addition, the nucleic acid(s) carry at the 5′ end a means for attaching the nucleic acid(s) to a solid support; (2) mixing said nucleic acid template(s) with one or more colony primers X, which can hybridize to the oligonucleotide sequence Z and carries at the 5′ end a means for attaching the colony primers to a solid support, in the presence of a solid support so that the 5′ ends of both the nucleic acid template and the colony primers bind to the solid support; (3) performing one or more nucleic acid amplification reactions on the bound template(s), so that nucleic acid colonies are generated and optionally, performing at least one step of sequence determination of one or more of the nucleic acid colonies generated. Solid supports, kits and apparatus for use in these methods.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/506,146, now U.S. Pat. No. 8,652,810, which is a DivisionalApplication of U.S. application Ser. No. 09/806,531 filed Oct. 19, 2001,now U.S. Pat. No. 7,115,400, which in turn is the national stage under35 U.S.C. of international application PCT/GB99/03248, filed Sep. 30,1999, which designated the United States and was published in English.The present application also claims the benefit of United Kingdom App.No. 98307985.6, filed Sep. 30, 1998. The disclosure of each of theabove-identified applications is incorporated herein by reference in itsentirety.

This invention relates to the field of nucleic acid amplification andsequencing. More specifically, this invention relates to nucleic acidamplification and sequencing methods, and apparatus and kits useful forlarge scale high throughput amplification and sequencing of nucleicacids.

Nucleic acid sequence analysis has become a corner-stone in manyactivities in biology, biotechnology and medicine. The ability todetermine nucleic acid sequences has become increasingly important asefforts have commenced to determine the sequences of the large genomesof humans and other higher organisms and also, for example, in singlenucleotide polymorphism detection and screening and gene expressionmonitoring. The genetic information provided by nucleic acid sequencinghas many applications in areas such as for example drug target discoveryand validation, disease diagnosis and risk scoring and organismidentification and characterization.

The first step in such applications is the determination of the actualchemical composition of the nucleic acids of interest, more preciselythe determination of the sequence of occurrence of the four basesadenine (A), cytosine (C), guanine (G) and thymine (T) or uracil (U)which comprise nucleic acids. However, such applications require thesequencing of nucleic acids on a large scale, making high throughputmethods of nucleic acid sequencing extremely desirable.

Methods of nucleic acid sequencing are documented in the art. The twomost commonly used are the chemical cleavage technique by Maxam andGilbert which relies on base-specific chemistry and the now more popularSanger sequencing technique which relies on an enzymatic chainterminating principle and is now used on a routine basis for nucleicacid sequencing.

In Sanger sequencing, each nucleic acid to be sequenced is replicated ina reaction involving DNA polymerase, deoxynucleotide triphosphates(dNTPs) and dideoxynucleotide triphosphates (ddNTPs). The DNA polymerasecan incorporate both dNTPs and ddNTPs into the growing DNA strand.However, once a ddNTP is incorporated, the 3′ end of the growing DNAstrand lacks a hydroxyl group and is no longer a substrate for chainelongation, thus terminating the nucleic acid chain. Hence, in aparticular reaction including one type of ddNTP a mixture of nucleicacids of different lengths is produced, all terminating with the sameddNTP. Usually separate reactions are set up for each of the four typesof ddNTP and the distribution of lengths of the nucleic acid fragmentsproduced is analysed by denaturing gel electrophoresis (which resolvesnucleic acid fragments according to their size), or more recently, bymass-spectroscopy. Usually, one or more of the deoxynucleotidetriphosphates in the reaction mixture is labelled to enable detection ofthe fragments of different lengths.

The above described methods are disadvantageous because each nucleicacid to be sequenced has to be processed individually during thebiochemical reaction. Gel electrophoresis is cumbersome, labourintensive and intrinsically slow even when capillary electrophoresis isused and is not well suited for large scale high throughput sequencing.In addition, the subsequent determination of the sequence is cumbersome.Mass-spectroscopy is still at the prototype level, requires veryexpensive apparatus and each sample has to be analysed individually.

One way to increase throughput is to process many samples in parallel.Methods using DNA hybridization of nucleic acid probes are in use andallow for some multiplexing of the process during the biochemical andthe electrophoretic processes, but at the cost of lengthy additionalmanipulations.

More recently methods based on DNA chips and DNA hybridization arebecoming available (Thomas and Burke Exp. Opin. Ther. Patents 8: 503-508(1998)). These methods are disadvantageous because for each application,a DNA chip has to be designed and manufactured first: this is a lengthyoperation and the price of an individual chip drops only when very largenumbers of the chip are required. Also, the chips are not reusable andfor each chip only one sample of nucleic acids, e.g. one patient to bediagnosed, can be processed at each time. Finally, the extent ofsequence which can be analysed by such a chip is limited to less than100,000 bases, and is limited to some applications such as DNAgenotyping and gene expression profiling.

In most known techniques for nucleic acid sequence analysis,amplification of the nucleic acids of interest is a prerequisite step inorder to obtain the nucleic acid in a quantity sufficient for analysis.

Several methods of nucleic acid amplification are well known anddocumented in the art. For example, nucleic acids can be amplified byinserting the nucleic acid of interest into an expression vectorconstruct. Such vectors can then be introduced into suitable biologicalhost cells and the vector DNA, including the nucleic acid of interest,amplified by culturing the biological host using well establishedprotocols.

Nucleic acids amplified by such methods can be isolated from the hostcells by methods well known and documented in the art. However, suchmethods have the disadvantage of being generally time consuming, labourintensive and difficult to automate.

The technique of DNA amplification by the polymerase chain reaction(PCR) was disclosed in 1985 (Saiki et al. Science 230, 1350-1354) and isnow a method well known and documented in the art. A target nucleic acidfragment of interest can be amplified using two short oligonucleotidesequences (usually referred to as primers) which are specific to knownsequences flanking the DNA sequence that is to be amplified. The primershybridize to opposite strands of the double-stranded DNA fragment afterit has been denatured, and are oriented so that DNA synthesis by the DNApolymerase proceeds through the region between the two primers, with theprimer sequences being extended by the sequential incorporation ofnucleotides by the polymerase. The extension reactions create twodouble-stranded target regions, each of which can again be denaturedready for a second cycle of hybridisation and extension. The third cycleproduces two double-stranded molecules that comprise precisely thetarget region in double-stranded form. By repeated cycles of heatdenaturation, primer hybridisation, and extension, there follows a rapidexponential accumulation of the specific target fragment of DNA.Traditionally, this method is performed in solution and the amplifiedtarget nucleic acid fragment purified from solution by methods wellknown in the art, for example by gel electrophoresis.

More recently, however, methods have been disclosed which use one primergrafted to a surface in conjunction with free primers in solution. Thesemethods allow the simultaneous amplification and attachment of a PCRproduct onto the surface (Oroskar, A. A. et al., Clinical Chemistry42:1547 (1996)).

WO96/04404 (Mosaic Technologies, Inc. et al) discloses a method ofdetection of a target nucleic acid in a sample which potentiallycontains the target nucleic acid. The method involves the induction of aPCR based amplification of the target nucleic acid only when the targetnucleic acid is present in the sample being tested. For theamplification of the target sequence, both primers are attached to asolid support, which results in the amplified target nucleic acidsequences also being attached to the solid support. The amplificationtechnique disclosed in this document is sometimes referred to as the“bridge amplification” technique. In this technique the two primers are,as for conventional PCR, specifically designed so that they flank theparticular target DNA sequence to be amplified. Thus, if the particulartarget nucleic acid is present in the sample, the target nucleic acidcan hybridise to the primers and be amplified by PCR. The first step inthis PCR amplification process is the hybridisation of the targetnucleic acid to the first specific primer attached to the support(“primer 1”). A first amplification product, which is complementary tothe target nucleic acid, is then formed by extension of the primer 1sequence. On subjecting the support to denaturation conditions thetarget nucleic acid is released and can then participate in furtherhybridisation reactions with other primer 1 sequences which may beattached to the support. The first amplification product which isattached to the support, may then hybridise with the second specificprimer (“primer 2”) attached to the support and a second amplificationproduct comprising a nucleic acid sequence complementary to the firstamplification product can be formed by extension of the primer 2sequence and is also attached to the support. Thus, the target nucleicacid and the first and second amplification products are capable ofparticipating in a plurality of hybridisation and extension processes,limited only by the initial presence of the target nucleic acid and thenumber of primer 1 and primer 2 sequences initially present and theresult is a number of copies of the target sequence attached to thesurface.

Since, on carrying out this process, amplification products are onlyformed if the target nucleic acid is present, monitoring the support forthe presence or absence of one or more amplification products isindicative of the presence or absence of a specific target sequence.

The Mosaic technique can be used to achieve an amount of multiplexing inthat several different target nucleic acid sequences can be amplifiedsimultaneously by arraying different sets of first and second primers,specific for each different target nucleic acid sequence, on differentregions of the solid support.

The disadvantage of the Mosaic process is that, as the first and secondprimer sequences have to be specific for each target nucleic acid to beamplified, it can only be used to amplify known sequences. In addition,the throughput is limited by the number of different sets of specificprimers and subsequently amplified target nucleic acid molecules whichcan be arrayed in distinct regions of a given solid support and the timetaken to array the nucleic acids in distinct regions. Also, the Mosaicprocess requires that 2 different primers are homogeneously attached bythe 5′ end to the support within the distinct region where theamplification product is formed. This cannot be achieved with presentlyavailable DNA chip manufacturing technology and has to be achieved bysome means of sample dispensing. Thus, the density that can be achievedby this approach has the same limitation as other classical arrayingtechnologies. A further limitation is the speed of monitoring theindividual distinct regions of the support for the presence or absenceof the amplified target nucleic acids.

Arraying of DNA samples is classically performed on membranes (e.g.,nylon or nitro-cellulose membranes). The use of suitable robotics (e.g.,Q-bot™, Genetix Ltd, Dorset BH23 3TG UK) means that it is possible toobtain a density of up to 10 samples/mm². In such methods, the DNA iscovalently linked to a membrane by physicochemical means (e.g., UVirradiation) and the arraying of large DNA molecules (e.g. moleculesover 100 nucleotides long) as well as smaller DNA molecules such asoligonucleotide primers is possible.

Other techniques are known whereby higher density arrays ofoligonucleotides can be obtained. For example, approaches based onpre-arrayed glass slides wherein arrays of reactive areas are obtainedby ink-jet technology (Blanchard, A. P. and L. Hood, Microbial andComparative Genomics, 1:225 (1996)) or arrays of reactive polyacrylamidegels (Yershov, G. et al., Proceedings of the National Academy ofScience, USA, 93:4913-4918 (1996)) allow in theory the arraying of up to100 samples/mm².

Higher sample densities still are achievable by the use of DNA chips(Fodor, S. P. A. et al., Science 251:767 (1991)). Currently, chips with625 oligonucleotide probes/mm² are used in molecular biology techniques(Lockhart, D. J. et al., Nature Biotechnology 14:1675 (1996)). Probedensities of up to 250 000 samples/cm² (2500/mm²) are claimed to beachievable (Chee, M. et al., Science 274:610 (1996)). However, atpresent up to 132000 different oligonucleotides can be arrayed on asingle chips of approximately 2.5 cm². Importantly, these chips aremanufactured in such a way so that the 3′OH end of the oligonucleotideis attached to the solid surface. This means that oligonucleotidesattached to chips in such a way cannot be used as primers in a PCRamplification reaction.

Importantly, when PCR products are linked to the vessel in which PCRamplification takes place, the density of the resultant array of PCRproducts is limited by the available vessel. Currently available vesselsare only in 96 well microtiter plate format. These allow only around0.02 samples of PCR products/mm² of surface to be obtained.

For example, using the commercially available Nucleolink™ system (NuncA/S, Roskilde, Denmark) it is possible to achieve simultaneousamplification and arraying of samples at a density of 0.02 samples/mm²in wells on the surface of which oligonucleotide primers have beengrafted. However, technical problems mean that it is unlikely that asignificant increase in this sample density will be achieved with thisapproach.

Thus, it can be seen that in order to increase throughput there is aneed in the art for new methods of nucleic acid amplification whichallow the simultaneous amplification and array of nucleic acid samplesat a higher density, and furthermore, allows the monitoring of samplesat a faster rate, preferably in parallel.

In addition, it is apparent that there is a need in the art for newmethods of sequencing which allow large numbers of samples to beprocessed and sequenced in parallel, i.e. there is a need for methods ofsequencing which allow significant multiplexing of the process.Significant multiplexing of the sequencing process would in turn lead toa higher throughput than that achievable with the methods of sequencingknown in the art. Such new methods would be even more desirable if theycould achieve such high throughput sequencing at a reasonable cost andwith less labour intensiveness than conventional sequencing techniques.

The present invention describes new methods of solid-phase nucleic acidamplification which enable a large number of distinct nucleic acidsequences to be arrayed and amplified simultaneously and at a highdensity. The invention also describes methods by which a large number ofdistinct amplified nucleic acid sequences can be monitored at a fastrate and, if desired, in parallel. The invention also describes methodsby which the sequences of a large number of distinct nucleic acids canbe determined simultaneously and within a short period of time. Themethods are particularly useful in, but not limited to, the sequencingof a whole genome, or situations where many genes (e.g. 500) from manyindividuals (e.g. 500) have to be sequenced simultaneously, or thesimultaneous scoring of large numbers (e.g. millions) of polymorphisms,or the monitoring of the expression of a large number of genes (e.g.100,000) simultaneously.

The present invention therefore provides a method for amplification ofat least one nucleic acid comprising the following steps:—

(1) forming at least one nucleic acid template comprising the nucleicacid(s) to be amplified, wherein said nucleic acid(s) contains at the 5′end an oligonucleotide sequence Y and at the 3′ end an oligonucleotidesequence Z and, in addition, the nucleic acid(s) carry at the 5′ end ameans for attaching the nucleic acid(s) to a solid support;

(2) mixing said nucleic acid template(s) with one or more colony primersX, which can hybridize to the oligonucleotide sequence Z and carries atthe 5′ end a means for attaching the colony primers to a solid support,in the presence of a solid support so that the 5′ ends of both thenucleic acid template and the colony primers bind to the solid support;

(3) performing one or more nucleic acid amplification reactions on thebound template(s), so that nucleic acid colonies are generated.

In a further embodiment of the invention, two different colony primers Xare mixed with the nucleic acid template(s) in step (2) of the method.Preferably the sequences of colony primers X are such that theoligonucleotide sequence Z can hybridise to one of the colony primers Xand the oligonucleotide sequence Y is the same as one of the colonyprimers X.

In an alternative embodiment of the invention, the oligonucleotidesequence Z is complementary to oligonucleotide sequence Y, referred toas Y′ and colony primer X is of the same sequence as oligonucleotidesequence Y.

In a yet further embodiment of the invention, the colony primer X maycomprise a degenerate primer sequence and the nucleic acid template(s)comprise the nucleic acid(s) to be amplified and do not containoligonucleotide sequences Y or Z at the 5′ and 3′ ends respectively.

In a further aspect of the invention, the method comprises theadditional step of performing at least one step of sequencedetermination of one or more of the nucleic acid colonies generated instep (3).

Thus the invention also provides a method for sequencing of at least onenucleic acid comprising the following steps:—

(1) forming at least one nucleic acid template comprising the nucleicacid(s) to be sequenced, wherein said nucleic acid(s) contains at the 5′end an oligonucleotide sequence Y and at the 3′ end an oligonucleotidesequence Z and, in addition, the nucleic acid(s) carry at the 5′ end ameans for attaching the nucleic acid(s) to a solid support;

(2) mixing said nucleic acid template(s) with one or more colony primersX, which can hybridize to the oligonucleotide sequence Z and carries atthe 5′ end a means for attaching the colony primers to a solid support,in the presence of a solid support so that the 5′ ends of both thenucleic acid template and the colony primers bind to the solid support;

(3) performing one or more nucleic acid amplification reactions on thebound template(s), so that nucleic acid colonies are generated; and

(4) performing at least one step of sequence determination of at leastone of the nucleic acid colonies generated.

In a further embodiment of the invention the 5′ ends of both the nucleicacid template(s) and the colony primers carry a means for attaching thenucleic acid sequences covalently to the solid support. Preferably thismeans for covalent attachment is a chemically modifiable functionalgroup, such as for example, a phosphate group, a carboxylic or aldehydemoiety, a thiol, a hydroxyl, a dimethoxyltrityl (DMT), or an aminogroup, preferably an amino group.

Nucleic acids which may be amplified according to the methods of theinvention include DNA, for example, genomic DNA, cDNA, recombinant DNAor any form of synthetic or modified DNA, RNA, mRNA or any form ofsynthetic or modified RNA. Said nucleic acids may vary in length and maybe fragments or smaller parts of larger nucleic acid molecules.Preferably the nucleic acid to be amplified is at least 50 base pairs inlength and more preferably 150 to 4000 base pairs in length. The nucleicacid to be amplified may have a known or unknown sequence and may be ina single or double-stranded form. The nucleic acid to be amplified maybe derived from any source.

“Nucleic acid template” as used herein refers to an entity whichcomprises the nucleic acid to be amplified or sequenced in asingle-stranded form. As outlined below the nucleic acid to be amplifiedor sequenced can also be provided in a double stranded form. Thus,“nucleic acid templates” of the invention may be single or doublestranded nucleic acids. The nucleic acid templates to be used in themethod of the invention can be of variable lengths. Preferably they areat least 50 base pairs in length and more preferably 150 to 4000 basepairs in length. The nucleotides making up the nucleic acid templatesmay be naturally occurring or non-naturally occurring nucleotides. Thenucleic acid templates of the invention not only comprise the nucleicacid to be amplified but may in addition contain at the 5′ and 3′ endshort oligonucleotide sequences. The oligonucleotide sequence containedat the 5′ end is referred to herein as Y. Oligonucleotide sequence Y isof a known sequence and can be of variable length. Oligonucleotidesequence Y for use in the methods of the present invention is preferablyat least five nucleotides in length, preferably between 5 and 100nucleotides in length and more preferably of approximately 20nucleotides in length. Naturally occurring or non-naturally occurringnucleotides may be present in the oligonucleotide sequence Y. Asindicated above, preferably the sequence of oligonucleotide Y is thesame as the sequence of colony primer X. The oligonucleotide sequencecontained at the 3′ end of the nucleic acid templates of the inventionis referred to herein as Z. Oligonucleotide sequence Z is of a knownsequence and can be of variable length. Oligonucleotide sequence Z foruse in the methods of the present invention is preferably at least fivenucleotides in length, preferably between 5 and 100 nucleotides inlength and more preferably of approximately 20 nucleotides in length.Naturally occurring or non-naturally occurring nucleotides may bepresent in the oligonucleotide sequence Z. Oligonucleotide sequence Z isdesigned so that it hybridises with one of the colony primers X andpreferably is designed so that it is complementary to oligonucleotidesequence Y, referred to herein as Y′. The oligonucleotide sequences Yand Z contained at the 5′ and 3′ ends respectively of a nucleic acidtemplate need not be located at the extreme ends of the template. Forexample although the oligonucleotide sequences Y and Z are preferablylocated at or near the 5′ and 3′ ends (or termini) respectively of thenucleic acid templates (for example within 0 to 100 nucleotides of the5′ and 3′ termini) they may be located further away (e.g. greater than100 nucleotides) from the 5′ or 3′ termini of the nucleic acid template.The oligonucleotide sequences Y and Z may therefore be located at anyposition within the nucleic acid template providing the sequences Y andZ are on either side, i.e. flank, the nucleic acid sequence which is tobe amplified.

“Nucleic acid template” as used herein also includes an entity whichcomprises the nucleic acid to be amplified or sequenced in adouble-stranded form. When the nucleic acid template is in adouble-stranded form, the oligonucleotide sequences Y and Z arecontained at the 5′ and 3′ ends respectively of one of the strands. Theother strand, due to the base pairing rules of DNA, is complementary tothe strand containing oligonucleotide sequences Y and Z and thuscontains an oligonucleotide sequence Z′ at the 5′ end and anoligonucleotide sequence Y′ at the 3′ end.

“Colony primer” as used herein refers to an entity which comprises anoligonucleotide sequence which is capable of hybridizing to acomplementary sequence and initiate a specific polymerase reaction. Thesequence comprising the colony primer is chosen such that it has maximalhybridising activity with its complementary sequence and very lownon-specific hybridising activity to any other sequence. The sequence tobe used as a colony primer can include any sequence, but preferablyincludes 5′-AGAAGGAGAAGGAAAGGGAAAGGG or 5′-CACCAACCCAAACCAACCCAAACC. Thecolony primer can be 5 to 100 bases in length, but preferably 15 to 25bases in length. Naturally occurring or non-naturally occurringnucleotides may be present in the primer. One or two different colonyprimers may be used to generate nucleic acid colonies in the methods ofthe present invention. The colony primers for use in the presentinvention may also include degenerate primer sequences.

“Degenerate primer sequences” as used herein refers to a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.Such degenerate primers thus do not require the presence ofoligonucleotide sequences Y or Z in the nucleic acid template(s) forhybridization to the template to occur, although the use of degenerateprimers to hybridise to a template containing the oligonucleotidesequences X or Y is not excluded. Clearly however, for use in theamplification methods of the present invention, the degenerate primersmust hybridise to nucleic acid sequences in the template at sites eitherside, or flanking, the nucleic acid sequence which is to be amplified.

“Solid support” as used herein refers to any solid surface to whichnucleic acids can be covalently attached, such as for example latexbeads, dextran beads, polystyrene, polypropylene surface, polyacrylamidegel, gold surfaces, glass surfaces and silicon wafers. Preferably thesolid support is a glass surface.

“Means for attaching nucleic acids to a solid support” as used hereinrefers to any chemical or non-chemical attachment method includingchemically-modifiable functional groups. “Attachment” relates toimmobilization of nucleic acid on solid supports by either a covalentattachment or via irreversible passive adsorption or via affinitybetween molecules (for example, immobilization on an avidin-coatedsurface by biotinylated molecules). The attachment must be of sufficientstrength that it cannot be removed by washing with water or aqueousbuffer under DNA-denaturing conditions.

“Chemically-modifiable functional group” as used herein refers to agroup such as for example, a phosphate group, a carboxylic or aldehydemoiety, a thiol, or an amino group.

“Nucleic acid colony” as used herein refers to a discrete areacomprising multiple copies of a nucleic acid strand. Multiple copies ofthe complementary strand to the nucleic acid strand may also be presentin the same colony. The multiple copies of the nucleic acid strandsmaking up the colonies are generally immobilised on a solid support andmay be in a single or double stranded form. The nucleic acid colonies ofthe invention can be generated in different sizes and densitiesdepending on the conditions used. The size of colonies is preferablyfrom 0.2 μm to 6 μm, more preferably from 0.3 μm to 4 μm. The density ofnucleic acid colonies for use in the method of the invention istypically 10,000/mm² to 100,000/mm². It is believed that higherdensities, for example, 100,000/mm² to 1,000,000/mm² and 1,000,000/mm²to 10,000,000/mm² may be achieved.

The methods of the invention can be used to generate nucleic acidcolonies. Thus, a further aspect of the invention provides one or morenucleic acid colonies. A nucleic acid colony of the invention may begenerated from a single immobilised nucleic acid template of theinvention. The method of the invention allows the simultaneousproduction of a number of such nucleic acid colonies, each of which maycontain different immobilised nucleic acid strands.

Thus, a yet further aspect of the invention provides a plurality ofnucleic acid templates comprising the nucleic acids to be amplified,wherein said nucleic acids contain at their 5′ ends an oligonucleotidesequence Y and at the 3′ end an oligonucleotide sequence Z and, inaddition, the nucleic acid(s) carry at the 5′ end a means for attachingthe nucleic acid(s) to a solid support. Preferably this plurality ofnucleic acid templates are mixed with a plurality of colony primers Xwhich can hybridize to the oligonucleotide sequence Z and carry at the5′ end a means for attaching the colony primers to a solid support.Preferably said plurality of nucleic acid templates and colony primersare covalently bound to a solid support.

In a further embodiment of the invention, pluralities of two differentcolony primers X are mixed with the plurality of nucleic acid templates.Preferably the sequences of colony primers X are such that theoligonucleotide sequence Z can hybridise to one of the colony primers Xand the oligonucleotide sequence Y is the same as the sequence of one ofthe colony primers X.

In an alternative embodiment, the oligonucleotide sequence Z iscomplementary to oligonucleotide sequence Y, (Y′) and the plurality ofcolony primers X are of the same sequence as oligonucleotide sequence Y.

In a yet further embodiment, the plurality of colony primers X maycomprise a degenerate primer sequence and the plurality of nucleic acidtemplates comprise the nucleic acids to be amplified and do not containoligonucleotide sequences Y or Z at the 5′ and 3′ ends respectively.

The nucleic acid templates of the invention may be prepared usingtechniques which are standard or conventional in the art. Generallythese will be based on genetic engineering techniques.

The nucleic acids to be amplified can be obtained using methods wellknown and documented in the art. For example, by obtaining a nucleicacid sample such as, total DNA, genomic DNA, cDNA, total RNA, mRNA etc.by methods well known and documented in the art and generating fragmentstherefrom by, for example, limited restriction enzyme digestion or bymechanical means.

Typically, the nucleic acid to be amplified is first obtained in doublestranded form. When the nucleic acid is provided in single strandedform, for example mRNA, it is first made into a double stranded form bymeans well known and documented in the art, for example, using oligo-dTprimers and reverse transcriptase and DNA polymerase. Once the nucleicacid to be amplified is obtained in double stranded form of appropriatelength, oligonucleotide sequences corresponding to the oligonucleotidesequences Y and Z are joined to each end, i.e. to both the 5′ and 3′ends of the nucleic acid sequence to form a nucleic acid template. Thiscan be done using methods which are well known and documented in theart, for example by ligation, or by inserting the nucleic acid to beamplified into a biological vector at a site which is flanked by theappropriate oligonucleotide sequences. Alternatively, if at least partof the sequence of the nucleic acid to be amplified is known, thenucleic acid template containing oligonucleotide sequences Y and Z atthe 5′ and 3′ ends respectively, may be generated by PCR usingappropriate PCR primers which include sequences specific to the nucleicacid to be amplified. Before attaching the nucleic acid template to thesolid support, it can be made into a single stranded form using methodswhich are well known and documented in the art, for example by heatingto approximately 94° C. and quickly cooling to 0° C. on ice.

The oligonucleotide sequence contained at the 5′ end of the nucleic acidcan be of any sequence and any length and is denoted herein as sequenceY. Suitable lengths and sequences of oligonucleotide can be selectedusing methods well known and documented in the art. For example theoligonucleotide sequences attached to each end of the nucleic acid to beamplified are normally relatively short nucleotide sequences of between5 and 100 nucleotides in length. The oligonucleotide sequence containedat the 3′ end of the nucleic acid can be of any sequence and any lengthand is denoted herein as sequence Z. Suitable lengths and sequences ofoligonucleotide can be selected using methods well known and documentedin the art. For example the oligonucleotide sequences contained at eachend of the nucleic acid to be amplified are normally relatively shortnucleotide sequences of between 5 and 100 nucleotides in length.

The sequence of the oligonucleotide sequence Z is such that it canhybridise to one of the colony primers X. Preferably, the sequence ofthe oligonucleotide sequence Y is such that it is the same as one of thecolony primers X. More preferably, the oligonucleotide sequence Z iscomplementary to oligonucleotide sequence Y (Y′) and the colony primersX are of the same sequence as oligonucleotide sequence Y.

The oligonucleotide sequences Y and Z of the invention may be preparedusing techniques which are standard or conventional in the art, or maybe purchased from commercial sources.

When producing the nucleic acid templates of the invention additionaldesirable sequences can be introduced by methods well known anddocumented in the art. Such additional sequences include, for example,restriction enzyme sites or certain nucleic acid tags to enableamplification products of a given nucleic acid template sequence to beidentified. Other desirable sequences include fold-back DNA sequences(which form hairpin loops or other secondary structures when renderedsingle-stranded), ‘control’ DNA sequences which direct protein/DNAinteractions, such as for example a promoter DNA sequence which isrecognised by a nucleic acid polymerase or an operator DNA sequencewhich is recognised by a DNA-binding protein.

If there are a plurality of nucleic acid sequences to be amplified thenthe attachment of oligonucleotides Y and Z can be carried out in thesame or different reaction.

Once a nucleic acid template has been prepared, it may be amplifiedbefore being used in the methods of the present invention. Suchamplification may be carried out using methods well known and documentedin the art, for example by inserting the template nucleic acid into anexpression vector and amplifying it in a suitable biological host, oramplifying it by PCR. This amplification step is not however essential,as the method of the invention allows multiple copies of the nucleicacid template to be produced in a nucleic acid colony generated from asingle copy of the nucleic acid template.

Preferably the 5′ end of the nucleic acid template prepared as describedabove is modified to carry a means for attaching the nucleic acidtemplates covalently to a solid support. Such a means can be, forexample, a chemically modifiable functional group, such as, for examplea phosphate group, a carboxylic or aldehyde moiety, a thiol, or an aminogroup. Most preferably, the thiol, phosphate or amino group is used for5′-modification of the nucleic acid.

The colony primers of the invention may be prepared using techniqueswhich are standard or conventional in the art. Generally, the colonyprimers of the invention will be synthetic oligonucleotides generated bymethods well known and documented in the art or may be purchased fromcommercial sources.

According to the method of the invention one or two different colonyprimers X, can be used to amplify any nucleic acid sequence. Thiscontrasts with and has an advantage over many of the amplificationmethods known in the art such as, for example, that disclosed in WO96/04404, where different specific primers must be designed for eachparticular nucleic acid sequence to be amplified.

Preferably the 5′ ends of colony primers X of the invention are modifiedto carry a means for attaching the colony primers covalently to thesolid support. Preferably this means for covalent attachment is achemically modifiable functional group as described above. If desired,the colony primers can be designed to include additional desiredsequences such as, for example, restriction endonuclease sites or othertypes of cleavage sites each as ribozyme cleavage sites. Other desirablesequences include fold-back DNA sequences (which form hairpin loops orother secondary structures when rendered single-stranded), ‘control’ DNAsequences which direct a protein/DNA interaction, such as for example apromoter DNA sequence which is recognised by a nucleic acid polymeraseor an operator DNA sequence which is recognised by a DNA-bindingprotein.

Immobilisation of a colony primer X to a support by the 5′ end leavesits 3′ end remote from the support such that the colony primer isavailable for chain extension by a polymerase once hybridisation with acomplementary oligonucleotide sequence contained at the 3′ end of thenucleic acid template has taken place.

Once both the nucleic acid templates and colony primers of the inventionhave been synthesised they are mixed together in appropriate proportionsso that when they are attached to the solid support an appropriatedensity of attached nucleic acid templates and colony primers isobtained. Preferably the proportion of colony primers in the mixture ishigher than the proportion of nucleic acid templates. Preferably theratio of colony primers to nucleic acid templates is such that when thecolony primers and nucleic acid templates are immobilised to the solidsupport a “lawn” of colony primers is formed comprising a plurality ofcolony primers being located at an approximately uniform density overthe whole or a defined area of the solid support, with one or morenucleic acid templates being immobilised individually at intervalswithin the lawn of colony primers.

The nucleic acid templates may be provided in single stranded form.However, they may also be provided totally or partly in double strandedform, either with one 5′ end or both 5′ ends modified so as to allowattachment to the support. In that case, after completion of theattachment process, one might want to separate strands by means known inthe art, e.g. by heating to 94° C., before washing the released strandsaway. It will be appreciated that in the case where both strands of thedouble stranded molecules have reacted with the surface and are bothattached, the result will be the same as in the case when only onestrand is attached and one amplification step has been performed. Inother words, in the case where both strands of a double strandedtemplate nucleic acid have been attached, both strands are necessarilyattached close to each other and are indistinguishable from the resultof attaching only one strand and performing one amplification step.Thus, single stranded and double stranded template nucleic acids mightbe used for providing template nucleic acids attached to the surface andsuitable for colony generation.

The distance between the individual colony primers and the individualnucleic acid templates (and hence the density of the colony primers andnucleic acid templates) can be controlled by altering the concentrationof colony primers and nucleic acid templates that are immobilised to thesupport. A preferred density of colony primers is at least 1 fmol/mm²,preferably at least 10 fmol/mm², more preferably between 30 to 60fmol/mm². The density of nucleic acid templates for use in the method ofthe invention is typically 10,000/mm² to 100,000/mm². It is believedthat higher densities, for example, 100,000/mm² to 1,000,000/mm² and1,000,000/mm² to 10,000,000/mm² may be achieved.

Controlling the density of attached nucleic acid templates and colonyprimers in turn allows the final density of nucleic acid colonies on thesurface of the support to be controlled. This is due to the fact thataccording to the method of the invention, one nucleic acid colony canresult from the attachment of one nucleic acid template, providing thecolony primers of the invention are present in a suitable location onthe solid support (see in more detail below). The density of nucleicacid molecules within a single colony can also be controlled bycontrolling the density of attached colony primers.

Once the colony primers and nucleic acid templates of the invention havebeen immobilised on the solid support at the appropriate density,nucleic acid colonies of the invention can then be generated by carryingout an appropriate number of cycles of amplification on the covalentlybound template nucleic acid so that each colony comprises multiplecopies of the original immobilised nucleic acid template and itscomplementary sequence. One cycle of amplification consists of the stepsof hybridisation, extension and denaturation and these steps aregenerally performed using reagents and conditions well known in the artfor PCR.

A typical amplification reaction comprises subjecting the solid supportand attached nucleic acid template and colony primers to conditionswhich induce primer hybridisation, for example subjecting them to atemperature of around 65° C. Under these conditions the oligonucleotidesequence Z at the 3′ end of the nucleic acid template will hybridise tothe immobilised colony primer X and in the presence of conditions andreagents to support primer extension, for example a temperature ofaround 72° C., the presence of a nucleic acid polymerase, for example, aDNA dependent DNA polymerase or a reverse transcriptase molecule (i.e.an RNA dependent DNA polymerase), or an RNA polymerase, plus a supply ofnucleoside triphosphate molecules or any other nucleotide precursors,for example modified nucleoside triphosphate molecules, the colonyprimer will be extended by the addition of nucleotides complementary tothe template nucleic acid sequence.

Examples of nucleic acid polymerases which can be used in the presentinvention are DNA polymerase (Klenow fragment, T4 DNA polymerase),heat-stable DNA polymerases from a variety of thermostable bacteria(such as Taq, VENT, Pfu, Tfl DNA polymerases) as well as theirgenetically modified derivatives (TaqGold, VENTexo, Pfu exo). Acombination of RNA polymerase and reverse transcriptase can also be usedto generate the amplification of a DNA colony. Preferably the nucleicacid polymerase used for colony primer extension is stable under PCRreaction conditions, i.e. repeated cycles of heating and cooling, and isstable at the denaturation temperature used, usually approximately 94°C. Preferably the DNA polymerase used is Taq DNA polymerase.

Preferably the nucleoside triphosphate molecules used aredeoxyribonucleotide triphosphates, for example dATP, dTTP, dCTP, dGTP,or are ribonucleoside triphosphates for example dATP, dUTP, dCTP, dGTP.The nucleoside triphosphate molecules may be naturally or non-naturallyoccurring.

After the hybridisation and extension steps, on subjecting the supportand attached nucleic acids to denaturation conditions two immobilisednucleic acids will be present, the first being the initial immobilisednucleic acid template and the second being a nucleic acid complementarythereto, extending from one of the immobilised colony primers X. Boththe original immobilised nucleic acid template and the immobilisedextended colony primer formed are then able to initiate further roundsof amplification on subjecting the support to further cycles ofhybridisation, extension and denaturation. Such further rounds ofamplification will result in a nucleic acid colony comprising multipleimmobilised copies of the template nucleic acid and its complementarysequence.

The initial immobilisation of the template nucleic acid means that thetemplate nucleic acid can only hybridise with colony primers located ata distance within the total length of the template nucleic acid. Thusthe boundary of the nucleic acid colony formed is limited to arelatively local area to the area in which the initial template nucleicacid was immobilised. Clearly, once more copies of the template moleculeand its complement have been synthesised by carrying out further roundsof amplification, ie. further rounds of hybridisation, extension anddenaturation, then the boundary of the nucleic acid colony beinggenerated will be able to be extended further, although the boundary ofthe colony formed is still limited to a relatively local area to thearea in which the initial nucleic acid template was immobilised.

A schematic representation of a method of nucleic acid colony generationaccording to an embodiment of the present invention is shown in FIG. 1.FIG. 1(a) shows a colony primer X of the invention (shown here as havingthe sequence ATT), and a nucleic acid template of the inventioncontaining at the 5′ end an oligonucleotide sequence Y, here shown asATT and at the 3′ end an oligonucleotide sequence Z, here shown as AAT,which can hybridise to the colony primer sequence X. In the schematicrepresentation the colony primer X and the oligonucleotide sequences Yand Z are shown as being of only three nucleotides in length. Inpractice however, it will be appreciated that longer sequences wouldnormally be used. The 5′ ends of both the colony primer and the nucleicacid template carry a means for attaching the nucleic acid to a solidsupport. This means is denoted in FIG. 1 as a black square. This meansof attaching may result in a covalent or a non-covalent attachment.

Only one colony primer X and one template nucleic acid are shown in FIG.1(a) for simplicity. However, in practice a plurality of colony primersX will be present with a plurality of nucleic acid templates. Theplurality of colony primers X may comprise two different colony primersX. However, for simplicity the schematic representation shown in FIG. 1shows only one type of colony primer X, with the sequence ATT. Theplurality of nucleic acid templates may comprise different nucleic acidsequences in the central portion between the oligonucleotides Y and Z,but contain the same oligonucleotide sequences Y and Z at the 5′ and 3′ends respectively. Only one species of nucleic acid template is shownfor simplicity in FIG. 1, in which a portion of the sequence in thecentral portion is shown as CGG.

In the presence of a solid support, the 5′ ends of both the nucleic acidtemplate and colony primer bind to the support. This is depicted in FIG.1(b). The support and the attached nucleic acid template and colonyprimers are then subjected to conditions which induce primerhybridisation. FIG. 1(c) shows a nucleic acid template that hashybridised to a colony primer. Such hybridisation is enabled by virtueof the fact that the oligonucleotide sequence Z at the 3′ end of thenucleic acid template can hybridise to the colony primer. In theschematic representation oligonucleotide sequence Z is shown to becomplementary to the colony primer, although in practice an exactcomplementary sequence is not essential, providing hybridisation canoccur under the conditions the nucleic acid templates and colony primersare subjected to.

FIG. 1(d) shows the stage of primer extension. Here, under appropriateconditions of temperature and in the presence of a DNA polymerase and asupply of nucleotide precursors, for example dATP, dTTP, dCTP and dGTP,the DNA polymerase extends the colony primer from its 3′ end using thenucleic acid template as a template. When primer extension is complete,see FIG. 1(e), it can be seen that a second immobilised nucleic acidstrand has been generated which is complementary to the initial nucleicacid template. On separating the two nucleic acid strands by, forexample heating, two immobilised nucleic acids will be present, thefirst being the initial immobilised nucleic acid template and the secondbeing a nucleic acid complementary thereto, extending from one of theimmobilised colony primers X, see FIG. 1(f).

Both the original immobilised nucleic acid template and the immobilisedextended colony primer formed are then able to hybridise to other colonyprimers present (depicted as colony primers 2 and 3 in FIG. 1(g)) andafter a further round of primer extension (FIG. 1(h)) and strandseparation (FIG. 1(i)), four single stranded immobilised strands areprovided. Two of these contain sequences corresponding to the originalnucleic acid template and two contain sequences complementary thereto.

Further rounds of amplification beyond those shown in FIG. 1 can becarried out to result in a nucleic acid colony comprising multipleimmobilised copies of the template nucleic acid and its complementarysequence.

It can thus be seen that the method of the present invention allows thegeneration of a nucleic acid colony from a single immobilised nucleicacid template and that the size of these colonies can be controlled byaltering the number of rounds of amplification that the nucleic acidtemplate is subjected to. Thus the number of nucleic acid coloniesformed on the surface of the solid support is dependent upon the numberof nucleic acid templates which are initially immobilised to thesupport, providing there is a sufficient number of immobilised colonyprimers within the locality of each immobilised nucleic acid template.It is for this reason that preferably the solid support to which thecolony primers and nucleic acid templates have been immobilisedcomprises a lawn of immobilised colony primers at an appropriate densitywith nucleic acid templates immobilised at intervals within the lawn ofprimers.

Such so called “autopatterning” of nucleic acid colonies has anadvantage over many methods of the prior art in that a higher density ofnucleic acid colonies can be obtained due to the fact that the densitycan be controlled by regulating the density at which the nucleic acidtemplates are originally immobilised. Such a method is thus not limitedby, for example, having specifically to array specific primers onparticular local areas of the support and then initiate colony formationby spotting a particular sample containing nucleic acid template on thesame local area of primer. The numbers of colonies that can be arrayedusing prior art methods, for example those disclosed in WO96/04404(Mosaic Technologies, Inc.) is thus limited by the density/spacing atwhich the specific primer areas can be arrayed in the initial step.

By being able to control the initial density of the nucleic acidtemplates and hence the density of the nucleic acid colonies resultingfrom the nucleic acid templates, together with being able to control thesize of the nucleic acid colonies formed and in addition the density ofthe nucleic acid templates within individual colonies, an optimumsituation can be reached wherein a high density of individual nucleicacid colonies can be produced on a solid support of a large enough sizeand containing a large enough number of amplified sequences to enablesubsequent analysis or monitoring to be performed on the nucleic acidcolonies.

Once nucleic acid colonies have been generated it may be desirable tocarry out an additional step such as for example colony visualisation orsequence determination (see later). Colony visualisation might forexample be required if it was necessary to screen the colonies generatedfor the presence or absence of for example the whole or part of aparticular nucleic acid fragment. In this case the colony or colonieswhich contain the particular nucleic acid fragment could be detected bydesigning a nucleic acid probe which specifically hybridises to thenucleic acid fragment of interest.

Such a nucleic acid probe is preferably labelled with a detectableentity such as a fluorescent group, a biotin containing entity (whichcan be detected by for example an incubation with streptavidin labelledwith a fluorescent group), a radiolabel (which can be incorporated intoa nucleic acid probe by methods well known and documented in the art anddetected by detecting radioactivity for example by incubation withscintillation fluid), or a dye or other staining agent.

Alternatively, such a nucleic acid probe may be unlabelled and designedto act as a primer for the incorporation of a number of labellednucleotides with a nucleic acid polymerase. Detection of theincorporated label and thus the nucleic acid colonies can then becarried out.

The nucleic acid colonies of the invention are then prepared forhybridisation. Such preparation involves the treatment of the coloniesso that all or part of the nucleic acid templates making up the coloniesis present in a single stranded form. This can be achieved for exampleby heat denaturation of any double stranded DNA in the colonies.Alternatively the colonies may be treated with a restrictionendonuclease specific for a double stranded form of a sequence in thetemplate nucleic acid. Thus the endonuclease may be specific for eithera sequence contained in the oligonucleotide sequences Y or Z or anothersequence present in the template nucleic acid. After digestion thecolonies are heated so that double stranded DNA molecules are separatedand the colonies are washed to remove the non-immobilised strands thusleaving attached single stranded DNA in the colonies.

After preparation of the colonies for hybridisation, the labelled orunlabelled probe is then added to the colonies under conditionsappropriate for the hybridisation of the probe with its specific DNAsequence. Such conditions may be determined by a person skilled in theart using known methods and will depend on for example the sequence ofthe probe.

The probe may then be removed by heat denaturation and, if desired, aprobe specific for a second nucleic acid may be hybridised and detected.These steps may be repeated as many times as is desired.

Labelled probes which are hybridised to nucleic acid colonies can thenbe detected using apparatus including an appropriate detection device. Apreferred detection system for fluorescent labels is a charge-coupleddevice (CCD) camera, which can optionally be coupled to a magnifyingdevice, for example a microscope. Using such technology it is possibleto simultaneously monitor many colonies in parallel. For example, usinga microscope with a CCD camera and a 10× or 20× objective it is possibleto observe colonies over a surface of between 1 mm² and 4 mm², whichcorresponds to monitoring between 10 000 and 200 000 colonies inparallel. Moreover, it is anticipated that this number will increasewith improved optics and larger chips.

An alternative method of monitoring the colonies generated is to scanthe surface covered with colonies. For example systems in which up to100 000 000 colonies could be arrayed simultaneously and monitored bytaking pictures with the CCD camera over the whole surface can be used.In this way, it can be seen that up to 100 000 000 colonies could bemonitored in a short time.

Any other devices allowing detection and preferably quantitation offluorescence on a surface may be used to monitor the nucleic acidcolonies of the invention. For example fluorescent imagers or confocalmicroscopes could be used.

If the labels are radioactive then a radioactivity detection systemwould be required.

In methods of the present invention wherein the additional step ofperforming at least one step of sequence determination of at least oneof the nucleic acid colonies generated is performed, said sequencedetermination may be carried out using any appropriate solid phasesequencing technique. For example, one technique of sequencedetermination that may be used in the present invention involveshybridising an appropriate primer, sometimes referred to herein as a“sequencing primer”, with the nucleic acid template to be sequenced,extending the primer and detecting the nucleotides used to extend theprimer. Preferably the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base by base in situ nucleic acid sequencing.

The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Anyappropriate detectable label may be used, for example a fluorophore,radiolabel etc. Preferably a fluorescent label is used. The same ordifferent labels may be used for each different type of nucleotide.Where the label is a fluorophore and the same labels are used for eachdifferent type of nucleotide, each nucleotide incorporation can providea cumulative increase in signal detected at a particular wavelength. Ifdifferent labels are used then these signals may be detected atdifferent appropriate wavelengths. If desired a mixture of labelled andunlabelled nucleotides are provided.

In order to allow the hybridisation of an appropriate sequencing primerto the nucleic acid template to be sequenced the nucleic acid templateshould normally be in a single stranded form. If the nucleic acidtemplates making up the nucleic acid colonies are present in a doublestranded form these can be processed to provide single stranded nucleicacid templates using methods well known in the art, for example bydenturation, cleavage etc.

The sequencing primers which are hybridised to the nucleic acid templateand used for primer extension are preferably short oligonucleotides, forexample of 15 to 25 nucleotides in length. The sequence of the primersis designed so that they hybridise to part of the nucleic acid templateto be sequenced, preferably under stringent conditions. The sequence ofthe primers used for sequencing may have the same or similar sequencesto that of the colony primers used to generate the nucleic acid coloniesof the invention. The sequencing primers may be provided in solution orin an immobilised form.

Once the sequencing primer has been annealed to the nucleic acidtemplate to be sequenced by subjecting the nucleic acid template andsequencing primer to appropriate conditions, determined by methods wellknown in the art, primer extension is carried out, for example using anucleic acid polymerase and a supply of nucleotides, at least some ofwhich are provided in a labelled form, and conditions suitable forprimer extension if a suitable nucleotide is provided. Examples ofnucleic acid polymerases and nucleotides which may be used are describedabove.

Preferably after each primer extension step a washing step is includedin order to remove unincorporated nucleotides which may interfere withsubsequent steps. Once the primer extension step has been carried outthe nucleic acid colony is monitored in order to determine whether alabelled nucleotide has been incorporated into an extended primer. Theprimer extension step may then be repeated in order to determine thenext and subsequent nucleotides incorporated into an extended primer.

Any device allowing detection and preferably quantitation of theappropriate label, for example fluorescence or radioactivity, may beused for sequence determination. If the label is fluorescent a CCDcamera optionally attached to a magnifying device (as described above),may be used. In fact the devices used for the sequence determiningaspects of the present invention may be the same as those describedabove for monitoring the amplified nucleic acid colonies.

The detection system is preferably used in combination with an analysissystem in order to determine the number and nature of the nucleotidesincorporated at each colony after each step of primer extension. Thisanalysis, which may be carried out immediately after each primerextension step, or later using recorded data, allows the sequence of thenucleic acid template within a given colony to be determined.

If the sequence being determined is unknown, the nucleotides applied toa given colony are usually applied in a chosen order which is thenrepeated throughout the analysis, for example dATP, dTTP, dCTP, dGTP. Ifhowever, the sequence being determined is known and is beingresequenced, for example to analyse whether or not small differences insequence from the known sequence are present, the sequencingdetermination process may be made quicker by adding the nucleotides ateach step in the appropriate order, chosen according to the knownsequence. Differences from the given sequence are thus detected by thelack of incorporation of certain nucleotides at particular stages ofprimer extension.

Thus it can be seen that full or partial sequences of the amplifiednucleic acid templates making up particular nucleic acid colonies may bedetermined using the methods of the present invention.

In a further embodiment of the present invention, the full or partialsequence of more than one nucleic acid can be determined by determiningthe full or partial sequence of the amplified nucleic acid templatespresent in more than one nucleic acid colony. Preferably a plurality ofsequences are determined simultaneously.

Carrying out sequence determination of nucleic acids using the method ofthe present invention has the advantage that it is likely to be veryreliable due to the fact that large numbers of each nucleic acid to besequenced are provided within each nucleic acid colony of the invention.If desired, further improvements in reliability can be obtained byproviding a plurality of nucleic acid colonies comprising the samenucleic acid template to be sequenced, then determining the sequence foreach of the plurality of colonies and comparing the sequences thusdetermined.

Preferably the attachment of the colony primer and nucleic acid templateto the solid support is thermostable at the temperature to which thesupport may be subjected to during the nucleic acid amplificationreaction, for example temperatures of up to approximately 100° C., forexample approximately 94° C. Preferably the attachment is covalent innature.

In a yet further embodiment of the invention the covalent binding of thecolony primers and nucleic acid template(s) to the solid support isinduced by a crosslinking agent such as for example1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),succinic anhydride, phenyldiisothiocyanate or maleic anhydride, or ahetero-bifunctional crosslinker such as for examplem-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl[4-iodoacethyl]aminobenzoate (SIAB), Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),N-y-maleimidobutyryloxy-succinimide ester (GMBS),Succinimidyl-4-[p-maleimidophenyl]butyrate (SMPB) and the sulfo(water-soluble) corresponding compounds. The preferred crosslinkingreagents for use in the present invention, are s-SIAB, s-MBS and EDC.

In a yet further embodiment of the invention the solid support has aderivatised surface. In a yet further embodiment the derivatised surfaceof the solid support is subsequently modified with bifunctionalcrosslinking groups to provide a functionalised surface, preferably withreactive crosslinking groups.

“Derivatised surface” as used herein refers to a surface which has beenmodified with chemically reactive groups, for example amino, thiol oracrylate groups.

“Functionalised surface” as used herein refers to a derivatised surfacewhich has been modified with specific functional groups, for example themaleic or succinic functional moieties.

In the method of the present invention, to be useful for certainapplications, the attachment of colony primers and nucleic acidtemplates to a solid support has to fulfill several requirements. Theideal attachment should not be affected by either the exposure to hightemperatures and the repeated heating/cooling cycles employed during thenucleic acid amplification procedure. Moreover the support should allowthe obtaining of a density of attached colony primers of at least 1fmol/mm², preferably at least 10 fmol/mm², more preferably between 30 to60 fmol/mm². The ideal support should have a uniformly flat surface withlow fluorescence background and should also be thermally stable(non-deformable). Solid supports, which allow the passive adsorption ofDNA, as in certain types of plastic and synthetic nitrocellulosemembranes, are not suitable. Finally, the solid support should bedisposable, thus should not be of a high cost.

For these reasons, although the solid support may be any solid surfaceto which nucleic acids can be attached, such as for example latex beads,dextran beads, polystyrene, polypropylene surface, polyacrylamide gel,gold surfaces, glass surfaces and silicon wafers, preferably the solidsupport is a glass surface and the attachment of nucleic acids theretois a covalent attachment.

The covalent binding of the colony primers and nucleic acid templates tothe solid support can be carried out using techniques which are knownand documented in the art. For example, epoxysilane-amino covalentlinkage of oligonucleotides on solid supports such as porous glass beadshas been widely used for solid phase in situ synthesis ofoligonucleotides (via a 3′ end attachment) and has also been adapted for5′ end oligonucleotide attachment. Oligonucleotides modified at the 5′end with carboxylic or aldehyde moieties have been covalently attachedon hydrazine-derivatized latex beads (Kremsky et al 1987).

Other approaches for the attachment of oligonucleotides to solidsurfaces use crosslinkers, such as succinic anhydride,phenyldiisothiocyanate (Guo et al 1994), or maleic anhydride (Yang et al1998). Another widely used crosslinker is1-ethyl-3-(3-dimethylamonipropyl)-carbodiimide hydrochloride (EDC). EDCchemistry was first described by Gilham et al (1968) who attached DNAtemplates to paper (cellulose) via the 5′ end terminal phosphate group.Using EDC chemistry, other supports have been used such as, latex beads(Wolf et al 1987, Lund et al 1988), polystyrene microwells (Rasmussen etal 1991), controlled-pore glass (Ghosh et al 1987) and dextran molecules(Gingeras et al 1987). The condensation of 5′ amino-modifiedoligonucleotides with carbodiimide mediated reagent have been describedby Chu et al (1983), and by Egan et al (1982) for 5′ terminal phosphatemodification group.

The yield of oligonucleotide attachment via the 5′ termini usingcarbodiimides can reach 60%, but non-specific attachment via theinternal nucleotides of the oligonucleotide is a major drawback.Rasmussen et al (1991) have enhanced to 85% the specific attachment viathe 5′ end by derivatizing the surface using secondary amino groups.

More recent papers have reported the advantages of thehetero-bifunctional cross-linkers. Hetero- or mono-bifunctionalcross-linkers have been widely used to prepare peptide carrier conjugatemolecules (peptide-protein) in order to enhance immunogenicity inanimals (Peeters et al 1989). Most of these grafting reagents have beendescribed to form stable covalent links in aqueous solution. Thesecrosslinking reagents have been used to bind DNA onto a solid surface atonly one point of the molecule.

Chrisey et al (1996) have studied the efficiency and stability of DNAsolid phase attachment using 6 different hetero-bifunctionalcross-linkers. In this example, the attachment occurs only at the 5′ endof DNA oligomers modified by a thiol group. This type of attachment hasalso been described by O'Donnell-Maloney et al (1996) for the attachmentof DNA targets in a MALDI-TOF sequence analysis and by HamamatsuPhotonics F.K. company (EP-A-665293) for determining base sequence ofnucleic acid on a solid surface.

Very few studies concerning the thermal stability of the attachment ofthe oligonucleotides to the solid support have been done. Chrisey et al(1996) reported that with the Succinimidyl-4-[p-maleimidophenyl]butyrate(SMPB) cross-linker, almost 60% of molecules are released from the glasssurface during heat treatment. But the thermal stability of the otherreagents have not been described.

In order to generate nucleic acid colonies via the solid phaseamplification reaction as described in the present application, colonyprimers and nucleic acid templates need to be specifically attached attheir 5′ ends to the solid surface, preferably glass. Briefly, the glasssurface can be derivatized with reactive amino groups by silanizationusing amino-alkoxy silanes. Suitable silane reagents includeaminopropyltrimethoxy-silane, aminopropyltriethoxysilane and4-aminobutyltriethoxysilane. Glass surfaces can also be derivatized withother reactive groups, such as acrylate or epoxy using epoxysilane,acrylatesilane and acrylamidesilane. Following the derivatization step,nucleic acid molecules (colony primers or nucleic acid templates) havinga chemically modifiable functional group at their 5′ end, for examplephosphate, thiol or amino groups are covalently attached to thederivatized surface by a crosslinking reagent such as those describedabove.

Alternatively, the derivatization step can be followed by attaching abifunctional cross-linking reagent to the surface amino groups therebyproviding a modified functionalized surface. Nucleic acid molecules(colony primers or nucleic acid templates) having 5′-phosphate, thiol oramino groups are then reacted with the functionalized surface forming acovalent linkage between the nucleic acid and the glass.

Potential cross-linking and grafting reagents that can be used forcovalent DNA/oligonucleotide grafting on the solid support includesuccinic anhydride, (1-ethyl-3[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC), m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS), N-succinimidyl[4-iodoacethyl]aminobenzoate (SIAB), Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),N-y-maleimidobutyryloxy-succinimide ester (GMBS),Succinimidyl-4-[p-maleimidophenyl]butyrate (SMPB) and the sulfo(water-soluble) corresponding compounds. The preferred crosslinkingreagents, according to the present invention, are s-SIAB, s-MBS and EDC.s-MBS is a maleimide-succinimide hetero-bifunctional cross-linker ands-SIAB is an iodoacethyl-succinimide hetero-bifunctional cross-linker.Both are capable of forming a covalent bond respectively with SH groupsand primary amino groups. EDC is a carbodiimide-reagent that mediatescovalent attachment of phosphate and amino groups.

The colony primers and nucleic acid templates are generally modified atthe 5′ end by a phosphate group or by a primary amino group (for EDCgrafting reagent) or a thiol group (for s-SIAB or s-MBS linkers).

Thus, a further aspect of the invention provides a solid support, towhich there is attached a plurality of colony primers X as describedabove and at least one nucleic acid template as described above, whereinsaid nucleic acid templates contain at their 5′ ends an oligonucleotidesequence Y as described above, and at their 3′ ends an oligonucleotidesequence Z as described above. Preferably a plurality of nucleic acidtemplates are attached to said solid support, which is preferably glass.Preferably the attachment of the nucleic acid templates and colonyprimers to the solid support is covalent. By performing one or morerounds of nucleic acid amplification on the immobilised nucleic acidtemplate(s) using methods as described above, nucleic acid colonies ofthe invention may be formed. A yet further aspect of the invention is,therefore, a support comprising one or more nucleic acid colonies of theinvention. A yet further aspect of the invention provides the use of thesolid supports of the invention in methods of nucleic acid amplificationor sequencing. Such methods of nucleic acid amplification or sequencinginclude the methods of the present invention.

A yet further aspect of the invention provides the use of a derivatizedor functionalized support, prepared as described above in methods ofnucleic acid amplification or sequencing. Such methods of nucleic acidamplification or sequencing include the methods of the presentinvention.

A yet further aspect of the invention provides an apparatus for carryingout the methods of the invention or an apparatus for producing a solidsupport comprising nucleic acid colonies of the invention. Suchapparatus might comprise for example a plurality of nucleic acidtemplates and colony primers of the invention bound, preferablycovalently, to a solid support as outlined above, together with anucleic acid polymerase, a plurality of nucleotide precursors such asthose described above, a proportion of which may be labelled, and ameans for controlling temperature. Alternatively, the apparatus mightcomprise for example a support comprising one or more nucleic acidcolonies of the invention. Preferably the apparatus also comprises adetecting means for detecting and distinguishing signals from individualnucleic acid colonies arrayed on the solid support according to themethods of the present invention. For example such a detecting meansmight comprise a charge-coupled device operatively connected to amagnifying device such as a microscope as described above.

Preferably any apparatuses of the invention are provided in an automatedform.

The present application provides a solution to current and emergingneeds that scientists and the biotechnology industry are trying toaddress in the fields of genomics, pharmacogenomics, drug discovery,food characterization and genotyping. Thus the method of the presentinvention has potential application in for example: nucleic acidsequencing and re-sequencing, diagnostics and screening, gene expressionmonitoring, genetic diversity profiling, whole genome polymorphismdiscovery and scoring, the creation of genome slides (whole genome of apatient on a microscope slide) and whole genome sequencing.

Thus the present invention may be used to carry out nucleic acidsequencing and re-sequencing, where for example a selected number ofgenes are specifically amplified into colonies for complete DNAsequencing. Gene re-sequencing allows the identification of all known ornovel genetic polymorphisms of the investigated genes. Applications arein medical diagnosis and genetic identification of living organisms.

For use of the present invention in diagnostics and screening, wholegenomes or fractions of genomes may be amplified into colonies for DNAsequencing of known single nucleotide polymorphisms (SNP). SNPidentification has application in medical genetic research to identifygenetic risk factors associated with diseases. SNP genotyping will alsohave diagnostic applications in pharmaco-genomics for the identificationand treatment of patients with specific medications.

For use of the present invention in genetic diversity profiling,populations of for example organisms or cells or tissues can beidentified by the amplification of the sample DNA into colonies,followed by the DNA sequencing of the specific “tags” for eachindividual genetic entity. In this way, the genetic diversity of thesample can be defined by counting the number of tags from eachindividual entity.

For use of the present invention in gene expression monitoring, theexpressed mRNA molecules of a tissue or organism under investigation areconverted into cDNA molecules which are amplified into sets of coloniesfor DNA sequencing. The frequency of colonies coding for a given mRNA isproportional to the frequency of the mRNA molecules present in thestarting tissue. Applications of gene expression monitoring are inbio-medical research.

A whole genome slide, where the entire genome of a living organism isrepresented in a number of DNA colonies numerous enough to comprise allthe sequences of that genome may be prepared using the methods of theinvention. The genome slide is the genetic card of any living organism.Genetic cards have applications in medical research and geneticidentification of living organisms of industrial value.

The present invention may also be used to carry out whole genomesequencing where the entire genome of a living organism is amplified assets of colonies for extensive DNA sequencing. Whole genome sequencingallows for example, 1) a precise identification of the genetic strain ofany living organism; 2) to discover novel genes encoded within thegenome and 3) to discover novel genetic polymorphisms.

The applications of the present invention are not limited to an analysisof nucleic acid samples from a single organism/patient. For example,nucleic acid tags can be incorporated into the nucleic acid templatesand amplified, and different nucleic acid tags can be used for eachorganism/patient. Thus, when the sequence of the amplified nucleic acidis determined, the sequence of the tag may also be determined and theorigin of the sample identified.

Thus, a further aspect of the invention provides the use of the methodsof the invention, or the nucleic acid colonies of the invention, or theplurality of nucleic acid templates of the invention, or the solidsupports of the invention, for providing nucleic acid molecules forsequencing and re-sequencing, gene expression monitoring, geneticdiversity profiling, diagnosis, screening, whole genome sequencing,whole genome polymorphism discovery and scoring and the preparation ofwhole genome slides (ie. the whole genome of an individual on onesupport), or any other applications involving the amplification ofnucleic acids or the sequencing thereof.

A yet further aspect of the invention provides a kit for use insequencing, re-sequencing, gene expression monitoring, genetic diversityprofiling, diagnosis, screening, whole genome sequencing, whole genomepolymorphism discovery and scoring, or any other applications involvingthe amplification of nucleic acids or the sequencing thereof. This kitcomprises a plurality of nucleic acid templates and colony primers ofthe invention bound to a solid support, as outlined above.

The invention will now be described in more detail in the followingnon-limiting Examples with reference to the following drawings in which:

FIG. 1: shows a schematic representation of a method of nucleic acidcolony generation according to an embodiment of the invention.

FIG. 2: Schematic representation of template preparation and subsequentattachment to the solid surface. In FIG. 2a the preparation of TemplatesA, B and B′ containing colony primer sequences is shown. The 3.2 Kbtemplate is generated from genomic DNA using PCR primers TP1 and TP2.Templates A (854 bp) end B (927 bp) are generated using PCR primersTPA1/TPA2 or TPB1/TPB2, respectively. The TPA1 and TPB1 oligonucleotidesare modified at their 5′-termini with either a phosphate or thiol groupfor subsequent chemical attachment (*). Note that the templates.obtained contain sequences corresponding to colony primers CP1 and/orCP2. The 11 exons of the gene are reported as “E1 to E11”. In FIG. 2bthe chemical attachment of colony primers and templates to glass surfaceis shown. Derivatization by ATS (aminopropyltriethoxysilane) isexemplified.

FIG. 3: DNA colonies generated from a colony primer. It shows the numberof colonies observed per 20× field as a function of the concentration oftemplate bound to the well. The lowest concentration of detectabletemplate corresponds to 10⁻¹³ M.

FIG. 4: Representation of discrimination between colonies originatedfrom two different templates. FIG. 4a shows the images of colonies madefrom both templates and negative controls. FIG. 4b shows the coloniesfrom both templates at the same position in the same well visualisedwith two different colours and negative controls. FIG. 4c shows thecoordinates of both colony types in a sub-section of a microscopy field.FIG. 4c demonstrates that colonies from different templates do notcoincide.

FIG. 5: Reaction schemes of the template or oligonucleotide attachmenton glass. Step A is the derivatization of the surface: glass slide aretreated with acidic solution to enhance free hydroxyl group on thesurface. The pretreated slides are immersed into a solution ofaminosilane. ATS: Aminopropyl triethoxysilane. Step B: B1 or B2 is thefunctionalization of glass surface with cross-linkers followed byoligonucleotide attachment. Amino group reacts with a cross linkingagent via an amide bond: step B1; s-MBS (sulfom-maleimidobenzoyl-N-hydroxysuccinimide ester) step B2; s-SIAB (sulfoN-succinimidyl[4-iodoacethyl]aminobenzoate). The oligonucleotides (5′end thiol modified oligonucleotide) are attached to the surface viaformation of a covalent bound between the thiol and the double bond ofthe maleimide. Phosphate buffered saline: (PBS, 0.1 M NaH₂PO₄, pH:6.5,0.15 M NaCl). B3: Attachment of oligonucleotides using EDC andImidazole. 5′ end phosphate of the modified oligonucleotides reacts withimidazole in the presence of EDC to give 5′-phosphor-imidazolidederivatives (not shown). The derivatives form a phosphoramidate bondwith amino groups of the derivatized glass surface. EDC:1-ethyl-3-(3-dimethylamonipropyl)-carbodiimide hydrochloride.

FIG. 6: it shows the number of colonies observed per 20× field as afunction of the concentration of template bound to the well. DNAtemplate were bound at different concentration either via the mediatedcoupling reagent (EDC) on amino derivatized glass surface (A) or ons-MBS functionalized glass surface (B). Double strand DNA colonies weresubmitted to restriction enzyme and the recovered single strandshybridized with a complementary oligonucleotide, cy5 fluorescentlylabeled.

FIG. 7: shows an example of in situ sequencing from DNA coloniesgenerated on glass. FIG. 7A shows the result after incubation withCy5™-dCTP on a sample that has not been incubated with primer p181. Onewill appreciate only 5 blurry spots can be observed, indicating that nodramatic spurious effect is taking place (such as Cy5™-dCTP aggregateprecipitation, adsorption or simply non specific incorporation to theDNA in the colonies or on the surface). FIG. 7B shows the result afterincubation with Cy5™-dUTP on a sample that has been incubated withprimer p181. One will appreciate that no fluorescent spot can beobserved, indicating that the incorporation of a fluorescent base cannottake place in detectable amounts when the nucleotide proposed forincorporation does not correspond to the sequence of the templatefollowing the hybridized primer. FIG. 7C shows the result afterincubation with Cy5™-dCTP on a sample that has been incubated withprimer p181. One will appreciate that many fluorescent spots can beobserved, indicating that the incorporation of a fluorescent base canindeed take place in detectable amounts when the nucleotide proposed forincorporation does correspond to the sequence of the template followingthe hybridized primer.

FIG. 8: shows hybridization of probes to oligonucleotides attached toNucleolink, before and after PCR cycling. The figure shows R58hybridization to CP2 (5′-(phosphate)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG)closed circles, CP8(5′(amino-hexamethylene)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG) closedtriangles, CP9 (5′(hydroxyl)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG)diamonds, CP10 (5′(dimethoxytrityl)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG)open circles and CP11 (5′(biotin)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG)open triangles.

EXAMPLES Example 1 Preparation of DNA Templates Suitable for theGeneration of DNA Colonies

DNA colonies have been generated from DNA templates and colony primers.The term “colony primer sequence” as used herein refers to a sequencecorresponding to the sequence of a colony primer and is elsewheresometimes referred to as “oligonucleotide sequence Y” or“oligonucleotide sequence Z′”.

The properties of the colony primers have been chosen based on aselection for oligonucleotide primers that show little non-specificnucleotide incorporation in the presence of heat-stable DNA polymerases.The colony primers, CPα (5′-p CACCAACCCAAACCAACCCAAACC) and CPβ (5′-pAGAAGGAGAAGGAAAGGGAAAGGG) have been selected due to their lowincorporation of radiolabeled (α³²P-dCTP] in the presence of a stableDNA polymerase (AmpliTaq, Perkin Elmer, Foster City, Calif.) in thestandard buffer and under thermocycling conditions (94° C. for 30seconds, 65° C. for 1 minute, 72° C. for 2 minutes, 50 cycles).

A 3.2 Kb DNA fragment was taken as a model system to demonstrate thefeasibility of colony generation using colony primers and DNA templates.The chosen template comprises the human gene for the receptor foradvanced glycosylation end-products (HUMOXRAGE, GenBank Acc. No.D28769). The RAGE-specific primers are depicted in Table 1. The 3.2 Kbtemplate was generated by PCR amplification from 0.1 μg human genomicDNA with 1 μM primers TP1 and TP2 with 1 unit of DNA polymerase(AmpliTaq, Perkin Elmer, Foster City, Calif.) in the standard buffer andunder thermocycling conditions (94° C. for 30 seconds, 65° C. for 1minute, 72° C. for 5 minutes, 40 cycles). This 3.2 Kb DNA fragment wasused as a template for secondary PCR to generate two shorter templatesfor colony generation (Templates A and B). The primers used to generatethe shorter templates contain both sequences specific to the templateand sequences of colony primers CP1 and CP2 to amplify the DNA on thesolid surface. In general, the PCR primer used to generate a DNAtemplate is modified at the 5′-terminus with either a phosphate or thiolmoiety. Thus after the PCR amplification, DNA fragments are generatedwhich contain the colony primer sequences at one or both terminiadjoining the RAGE DNA fragment of interest (see FIG. 2a ).

Template A (double stranded template containing the colony primersequence, CPP at both termini) was generated with 0.1 ng of the 3.2 Kbtemplate with 1 μM primers TPA1 and 1 μM TPA2 with 1 unit of DNApolymerase (AmpliTaq, Perkin Elmer, Foster City, Calif.) in the standardbuffer and under thermocycling conditions (94° C. for 30 seconds, 65° C.for 1 minute, 72° C. for 1 minutes, 30 cycles). The products were thenpurified over Qiagen Qia-quick columns (Qiagen GmbH, Hilden, Germany).

Template B (double stranded template which contains colony primersequences corresponding to CPβ) was generated with 0.1 ng of the 3.2 Kbtemplate with 1 μM primers TPB1 and 1 μM TPB2 with 1 unit of DNApolymerase (AmpliTaq, Perkin Elmer, Foster City, Calif.) in the standardbuffer and under thermocycling conditions (94° C. for 30 seconds, 65° C.for 1 minute, 72° C. for 1 minutes, 30 cycles). The products were thenpurified over Qiagen Qia-quick columns (Qiagen GmbH, Hilden, Germany).

Template B′ (double stranded template containing colony primer sequencescorresponding to CPα and CPβ at either end) was generated with 0.1 ng ofthe 3.2 Kb template with 1 μM primers TPB3 and 1 μM TPB4 with 1 unit of(AmpliTaq, Perkin Elmer, Foster City, Calif.) in the standard buffer andunder thermocycling conditions (94° C. for 30 seconds, 65° C. for 1minute, 72° C. for 1 minutes, 30 cycles). The products were thenpurified over Qiagen Qia-quick columns (Qiagen GmbH, Hilden, Germany).

All the specific oligonucleotides employed for the DNA templatespreparation and for the DNA colony generation have been reported in theTable 1 together with any chemical modification.

A general scheme showing the chemical attachment of colony primers andtemplates to the glass surface is reported in FIG. 2b , where thederivatization by ATS (aminopropyltriethoxysilane) is reported, as anon-limitative example.

TABLE 1  List of oligonucleotides used for templates preparationand colonies generation: Coordinates Oligonucleotide Name DNA sequence(orientation) Modification Use TP1 GAGGCCAGAACAGT 9810 (R) TemplateTCAAGG 3.2 Kb TP2 CCTGTGACAAGACG 6550 (F) Template ACTGAA 3.2 Kb CP1TTTTTTTTTTCACC None 5′P Generate AACCCAAACCAACC colonies CAAACC CP2TTTTTTTTTTAGAA None 5′P Generate GGAGAAGGAAAGGG colonies AAAGGG CP3TTTTTTTTTTCACC None 5′SH Generate AACCCAAACCAACC colonies CAAACC CP4TTTTTTTTTTAGAA None 5′SH Generate GGAGAAGGAAAGGG colonies AAAGGG CP5AGAAGGAGAAGGAA None 5′P Generate AGGGAAAGGGTTTT colonies TTTTTTTTTTTTNNCP6 AGAAGGAGAAGGAA None 5′P Generate AGGGAAAGGGGG colonies CP7TTTTTTTTTTCACC None 5′(NH₂) Generate AACCCAAACCAACC colonies CAAACC CP8TTTTTTTTTTAGAA None 5′(NH₂) Generate GGAGAAGGAAAGGG colonies AAAGGG CP9TTTTTTTTTTAGAA None 5′(OH) Control GGAGAAGGAAAGGG oligo AAAGGG CP10TTTTTTTTTTAGAA None 5′(DMT) Control GGAGAAGGAAAGGG oligo AAAGGG CP11TTTTTTTTTTAGAA None 5′(biotin) Control GGAGAAGGAAAGGG oligo AAAGGG TPA1AGAAGGAGAAGGAA 6550 (F) 5′P Template A AGGGAAAGGGCCTG TGACAAGACGACTG AATPA2 TTTTTTTTTTAGAA 7403 (R) 5′P Template A GGAGAAGGAAAGGGAAAGGGGCGGCCGC TGAGGCCAGTGGAA GTCAGA TPB3 TTTTTTTTTTCACC 9049 (F) NoneTemplate B′ AACCCAAACCAACC CAAACCGAGCTCAG GCTGAGGCAGGAGA ATTG TPB1AGAAGGAGAAGGAA 9265 (F) None Template B AGGGAAAGGGGAGC TGAGGAGGAAGAGA GGTPB2 AGAAGGAGAAGGAA 8411 (R) 5′P Template B AGGGAAAGGGGCGGCCGCTCGCCTGGTT CTGGAAGACA TPB4 AGAAGGAGAAGGAA 9265 (R) 5′SH Template B′AGGGAAAGGGGCGG CCGCTCGCCTGGTT CTGGAAGACA Coordinate from HUMOXRAGE geneAccession number D28769 (R) means “reverse” and (F) means “forward”

Example 1a Preparation of a Random DNA Template Flanked by a DegeneratePrimer

A 3.2 Kb DNA fragment was taken as a model system to demonstrate thefeasibility of colony generation from random primer PCR amplification.This strategy can be applied to sequencing of DNA fragments ofapproximately 100 Kb in length and, by combination of fragments to wholegenomes. A fragment of DNA of 3.2 Kb was generated by PCR from humangenomic DNA using PCR primers; TP1 5′-pGAGGCCAGAACAGTTCAAGG and TP25′-pCCTGTGACAAGACGACTGAA as described in example 1. The 3.2 Kb fragmentwas cut in smaller fragments by a combination of restriction enzymes(EcoRI and HhaI yielding 4 fragments of roughly 800 bp). The cut oruncut fragment DNAs were then mixed with the degenerate primer, p252(5′-P TTTTTTTTTTISISISISISIS, where I stands for inosine (which pairswith A, T and C) and S stands for G or C) and covalently coupled to theNucleolink wells (Nunc, Denmark). The tubes were then subjected torandom solid phase PCR amplification and visualized by hybridisationwith labeled DNA probes, as will be described in Example 2a.

Example 2 Covalent Binding of DNA Templates and Colony Primers on SolidSupport (Plastic) and Colony Formation with a Colony Primer

Covalent Binding of Template and Colony Primer to the Solid Support(Plastic)

A colony primer (CP2, 5′-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG),phosphorylated at its 5′ terminus (Microsynth GmBH, Switzerland), wasattached onto Nucleolink plastic microtitre wells (Nunc, Denmark) in thepresence of varying doses of Template A (prepared as described inexample 1). 8 wells were set up in duplicate with seven 1/10 dilutionsof template with CP2, starting with the highest concentration of 1 nM.

Microtitre wells, to which CP2 colony primer and the template arecovalently bound were prepared as follows. In each Nucleolink well, 30μl of a 1 μM solution of the colony primer with varying concentrationsof template diluted down from 1 nM in 10 mM 1-methyl-imidazole (pH 7.0)(Sigma Chemicals) was added. To each well, 10 μl of 40 mM1-ethyl-3-{3-dimethylaminopropyl)-carbodiimide (pH 7.0) (SigmaChemicals) in 10 mM 1-methyl-imidazole, was added to the solution ofcolony primer and template. The wells were then sealed and incubated at50° C. overnight. After the incubation, wells were rinsed twice with 200μl of RS (0.4 N NaOH, 0.25% Tween 20), incubated 15 minutes with 200 μlRS, washed twice with 200 μl RS and twice with 200 μl TNT (100 mMTrisHCl pH 7.5, 150 mM NaCl, 0.1% Tween 20). Tubes were dried at 50° C.and were stored in a sealed plastic bag at 4° C.

Colony Generation

Colony growing was initiated in each well with 20 μl of PCR mix; thefour dNTPs (0.2 mM), 0.1% BSA (bovine serum albumin), 0.1% Tween 20, 8%DMSO (dimethylsulfoxide, Fluka, Switzerland), 1×PCR buffer and 0.025units/μl of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, Calif.).The wells were then placed in the thermocycler and growing was performedby incubating the sealed wells 5 minutes at 94° C. and cycling for 50repetitions the following conditions: 94° C. for 30 seconds, 65° C. for2 minutes, 72° C. for 2 minutes. After completion of this program, thewells were kept at 8° C. until further use. Prior to hybridization wellsare filled with 50 μL TE (10 mM Tris, 1 mM EDTA, pH 7.4) heated at 94°C. for 5 minutes and chilled on ice before probe addition at 45° C.

Colonies Visualization

Probe:

The probe was a DNA fragment of 1405 base pairs comprising the sequenceof the template at their 3′ end (nucleotide positions 8405 to 9259). TheDNA probe was synthesized by PCR using two primers: p47(5′-GGCTAGGAGCTGAGGAGGAA), amplifying from base 8405, and TP2,biotinylated at 5′ end, amplifying from base 9876 of the antisensestrand.

Hybridization and Detection:

The probe was diluted to 1 nM in “easyhyb” (Boehringer-Mannheim,Germany) and 20 μL added to each well. The probe and the colonies weredenatured at 94° C. for 5 min and then incubated 6 hours at 50° C.Excess probes was washed at 50° C. in 2×SSC with 0.1% Tween. The DNAprobes were bound to avidin coated green fluorescence fluorospheres of adiameter of 0.04μ (Molecular Probes) in TNT for 1 hour at roomtemperature. Excess beads were washed with TNT. Colonies were visualizedby microscopy and image analysis as described in example 2a. FIG. 3shows the number of colonies observed per 20× field as a function of theconcentration of template bound to the well. The lowest concentration ofdetectable template corresponds to 10⁻¹³ M.

Example 2a Covalent Binding of DNA Templates and Colony Primers on SolidSupport (Plastic) and Colony Formation with a Degenerate Primer

Covalent Binding of Template and Colony Primer to the Solid Support(Plastic)

Microtitre wells with p252 and template DNA fragments were prepared asfollows:

In each Nucleolink well, 30 μl of a 1 μM solution of the colony primerp252 with varying concentrations of template diluted down from 0.5 nM in10 mM 1-methyl-imidazole (pH 7.0) (Sigma Chemicals) was added. To eachwell, 10 μl of 40 mM 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (pH7.0) (Sigma Chemicals) in 10 mM 1-methyl-imidazole, was added to thesolution of colony primer and template. The wells were then sealed andincubated at 50° C. overnight. After the incubation, wells were rinsedtwice with 200 μl of RS (0.4N NaOH, 0.25% Tween 20), incubated 15minutes with 200 μl RS, washed twice with 200 μl RS and twice with 200μl TNT (100 mM TrisHCl pH7.5, 150 mM NaCl, 0.1% Tween 20). Tubes weredried at 50° C. and were stored in a sealed plastic bag at 4° C.

Colony Generation

DNA colony generation was performed with a modified protocol to allowrandom priming in each well with 20 μl of PCR mix; the four dNTPs (0.2mM each), 0.1% BSA, 0.1% Tween 20, 8% DMSO (dimethylsulfoxide, Fluka,Switzerland), 1×PCR buffer and 0.025 units/Al of AmpliTaq DNA polymerase(Perkin Elmer, Foster City, Calif.). The wells were then placed in thethermocycler and amplification was performed by incubating the sealedwells 5 minutes at 94° C. and cycling for 50 repetitions the followingconditions: 94° C. for 30 seconds, 65° C. for 2 minutes, 72° C. for 2minutes. After completion of this program, the wells were kept at 8° C.until further use. Prior to hybridization wells are filled with 50 μL TE(10 mM Tris 1 mM EDTA pH 7.4) heated at 94° C. for 5 minutes and chilledon ice before probe addition at 45° C.

Colonies Visualization

Probes: Two DNA fragments of 546 and 1405 base pairs comprising thesequences of either extremities of the original template were amplifiedby PCR. The antisense strand of the probe was labeled with biotin,through the use of a 5′-biotinylated PCR primer. The base paircoordinates of the probes were 6550 to 7113 and 6734 to 9805.

Hybridization and detection: The probes were diluted to 1 nM in“easyhyb” (Boehringer-Mannheim, Germany) and 20 AL added to each well.The probe and the colonies were denatured at 94° C. for 5 min and thenincubated 6 hours at 50° C. Excess probes was washed at 50° C. in 2×SSCwith 0.1% tween. The DNA probes were bound to avidin coated greenfluorescence fluorospheres of a diameter of 40 nanometers (MolecularProbes, Portland Oreg.) in TNT for 1 hour at room temperature. Excessbeads were washed off with TNT. Fluorescence was detected using aninverted microscope (using the 20×/0.400 LD Achroplan objective, on theAxiovert S100TV, with an arc mercury lamp HBO 100 W/2, Carl Zeiss,Oberkochen, Germany) coupled to a 768(H)×512(V)pixel-CCD camera(Princeton Instruments Inc. Trenton, N.J., USA). Exposure were 20seconds through filter sets XF22 (Ex: 485DF22, Dichroic: 505DRLPO2 Em:530DF30) and XF47 (Ex: 640DF20, Dichroic: 670DRLPO2 Em: 682DF22) fromOmega Optical (Brattleboro Vt.) for FITC and Cy5 respectively. Data wereanalyzed using Winwiew software (Princeton Instruments Inc., TrentonN.J., USA). The numbers of colonies per field were counted in duplicatewells with image analysis software developed in house.

Example 3 Sequence Discrimination in Different Colonies Originated fromVarying Ratios of 2 Different Covalently Bound Templates and a ColonyPrimer

Covalent Binding of Templates and Colony Primer to the Solid Support(Plastic)

A colony primer (CP2: 5′pTTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG),phosphorylated at its 5′ termini (Microsynth GmbH, Switzerland), wasgrafted onto Nucleolink plastic microtitre wells (Nunc, Denmark) in thepresence of varying doses of the two templates A and B (prepared asdescribed in example 1). Series of 8 wells were set up in triplicatewith seven 1/10 dilutions of both templates starting with the highestconcentration of 1 nM. Template dilutions are set up in oppositedirections such that the highest concentration of one template coincideswith the lowest of the other.

Microtitre wells, to which CP2 primer and both templates are covalentlybound were prepared as follows. In each Nucleolink well, 30 μl of a 1 μMsolution of the CP2 primer with varying concentrations of both templatesdiluted down from 1 nM in 10 mM 1 methyl-imidazole (pH 7.0) (SigmaChemicals) were added. To each well, 10 μl of 40 mM1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (pH 7.0) (SigmaChemicals) in 10 mM 1-methyl-i imidazole (pH 7.0), was added to thesolution of colony primer and templates. The wells were then sealed andincubated at 50° C. for 4 hours. After the incubation, the wells wererinsed three times with 50 μl of RS (0.4 N NaOH, 0.25% Tween 20),incubated 15 minutes with 50 μl RS, washed three times with 50 μl RS andthree times with 50 μl TNT (100 mM TrisHCl pH 7.5, 150 mM NaCl, 0.1%Tween 20). Tubes were stored in TNT at 4° C.

Colonies Generation

Colony growing was initiated in each well with 20 μl of PCR mix; thefour dNTPs (0.2 mM), 0.1% BSA, 0.1% Tween 20, 8% DMSO(dimethylsulfoxide, Fluka, Switzerland), 1×PCR buffer and 0.025 units/μlof AmpliTaq DNA polymerase (Perkin Elmer, Foster City, Calif.).

The wells were then placed in the thermocycler and growing was performedby incubating the sealed wells 5 minutes at 94° C. and cycling for 50repetitions the following conditions: 94° C. for 30 seconds, 65° C. for5 minutes, 72° C. for 5 minutes. After completion of this program, thewells were kept at 8° C. until further use. Prior to hybridization wellsare filled with 50 μl TE (10 mM Tris, 1 mM EDTA, pH 7.4) heated at 94°C. for 5 minutes and chilled on ice before probe addition at 50° C.

Colonies Visualization

Probe: Two DNA fragments of 546 and 1405 base pairs corresponding to thesequences of the 3.2 Kb DNA fragment at the 5′- and 3′-termini wereamplified by PCR using one biotinylated primer (see example 2). The twoprobes were denatured by heating at 94° C. for 5 minutes, quick-chilledinto 1 M NaCl, 10 mM Tris pH 7.4 and allowed to bind to Strepatividincoated fluorospheres of diameter 0.04 μm labeled with different colorsfor 2 hours at 4° C. The probes bound to bead were diluted 20 fold in“easyhyb” solution prewarmed to 50° C. 20 μl of probes was added to eachwell containing denatured colonies.

Hybridization and detection: The hybridization was carried out at 50° C.for 5 hours. Excess probes was washed at 50° C. in 2×SSC with 0.1% SDS.Colonies were visualized by microscopy with a 20× objective, 20 secondexposure and image analysis as described in example 2a. FIG. 4a showsthe images of colonies made from both templates and negative controls.FIG. 4b shows the colonies from both templates at the same position inthe same well visualised with two different colours and negativecontrols. FIG. 4c shows the coordinates of both colony types in asub-section of a microscopy field. FIG. 4c demonstrates that coloniesfrom different templates do not coincide.

Example 4 Covalent Binding of DNA Templates and Oligonucleotides onGlass Solid Supports

Aminosilane-derivatized glass slides have been used as solid support tocovalently attach thiol-modified oligonucleotides probes usinghetero-bifunctional cross-linkers. The reagents selected havethiol-reactive (maleimide) and amino-reactive groups (succinimidylester). Oligonucleotide attachment yields and stability of theimmobilized molecules will be strongly dependent on the cross-linkerstability towards the conditions of the different treatments performed.The reaction schemes of the DNA templates or oligonucleotides attachmenton glass are described in FIG. 5.

The storage stability of glass slides prepared with the cross-linkerss-MBS and s-SIAB and its thermal stability have been evaluated. Animportant factor affecting the extent of hybridization of immobilizedoligonucleotide probes is the density of attached probes (Beattie etal., 1995; Joss et al., 1997). We have studied this effect by varyingthe concentration of oligonucleotides during the immobilization andassaying the density of attached oligos by hybridization.

Materials and Methods

Microscope glass slides acid pre-treatment—Microscope glass slides(Knittel, Merck ABS) were soaked in basic Helmanex solution during 1hour (HelmanexII^(R) 0.25%, 1N NaOH). The slides were rinsed with water,immersed overnight in 1N HCl, rinsed again in water and treated 1 hourin sulfuric acid solution (H₂SO₄/H₂O, 1/1, v/v, with a small amount offresh ammonium persulfate added). The slides were rinsed in water, inethanol and finally with pure acetone. Glass slides are dried and storedunder vacuum for further use.

Silanization of the surface—The pre-treated slides were immersed into a5% solution of ATS (aminopropyltriethoxysilane, Aldrich) in acetone.Silanization was carried out at room temperature for 2 hours. Afterthree washes in acetone (5 min/wash) the slides were rinsed once withethanol, dried and stored under vacuum.

Cross-linker attachment—Cross-linkers, s-MBS and s-SIAB (respectivelysulfo m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfoN-succinimidyl[4-iodoacethyl]aminobenzoate, Pierce, Rockford Ill.), areprepared as 20 mM solutions in PBS (phosphate-buffered saline, 0.1 MNaH₂PO₄, pH 7.2, 0.15 M NaCl). Silanized glass slides, on which 80 μL ofcross-linker solution was applied, were covered by a cleaned micro coverglass and reacted for 5 hours at 20° C. The glass slides were rinsed inPBS, briefly immersed in water and rinsed in ethanol. Slides were thendried and stored under vacuum in the dark for further use.

Oligonucleotide Attachment—Oligonucleotides were synthesized with 5′modifications of a thiol (CP3 and CP4 Eurogentec, Brussels) or aphosphate moiety (CP1 and CP2, Eurogentec, Brussels) using standardphosphoramidite chemistry.

-   -   5′-thiol oligonucleotide primers (CP3 and CP4) were prepared as        100 μM solutions in a saline phosphate buffer (NaPi: 0.1M        NaH₂PO₄ pH: 6.6, 0.15M NaCl) and drops of 1 μl applied on the        functionalized glass slide (functionalized with cross-linker)        for 5 hours at room temperature. Glass slides were kept under a        saturated wet atmosphere to avoid evaporation. Glass slides were        washed on a shaker in NaPi buffer. For thermal stability study        glass slides were immersed 2 times in Tris buffer (10 mM, pH 8)        for 5 min at 100° C. and directly immersed in 5×SSC (0.75 M        NaCl, 0.075 M NaCitrate pH 7) at 4° C. for 5 min. Slides were        stored in 5×SSC at 4° C. for further use.    -   5′-phosphate oligonucleotides primers (CP1 and CP2) were applied        (1 μl drops) for 5 hours at room temperature to        amino-derivatized glass as 1 μM solution in 10 mM        1-methyl-imidazole (pH 7.0) (Sigma Chemicals) containing 40 mM        of 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, Pierce,        Rockford Ill.). The slides were washed 2 times at 100° C. in        Tris buffer (10 mM, pH 8) and directly immersed in 5×SSC at        4° C. for 5 min. Slides were stored in 5×SSC at 4° C. for        further use.        Oligonucleotide and DNA Template Attachment

The 5′-thiol oligonucleotide primers (CP3 and CP4), and 5′-thioltemplate B′ were mixed in a saline phosphate buffer (NaPi: 0.1M NaH₂PO₄pH: 6.6, 0.15M NaCl). Concentration of DNA template varied from 0.001 to1 μM and from 0.1 to 100 μM for primers but were optimized at 1 μM and100 μM respectively for template and primers. The procedure describedabove for CP3 and CP4 attachment on functionalized glass surface wasthen followed.

The 5′-phosphate oligonucleotide primers (CP1 and CP2), and 5′-phosphatetemplate B were mixed in a 10 mM 1-methyl-imidazole (pH 7.0) (SigmaChemicals) solution containing 40 mM of1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, Pierce, RockfordIll.). Concentration of DNA template varied from 0.001 to 10 nM and from0.1 to 1 μM for primers, but were eventually optimized at 10 nM and 1 μMrespectively for template and primers. The procedure described above forCP1 and CP2 attachment on amino-derivatized glass surface was followed.

Hybridization with fluorescent probes—Oligonucleotide probes,fluorescently labeled with Cy5 or FITC at their 5′ end, were synthesizedby Eurogentec (Brussels). To prevent non-specific hybridization, glassslides were incubated with a blocking solution (5×SSC, Tween 0.1%, BSA0.1%) for 1 hour and washed on a shaker in 5×SSC (2 times, 5 min).Oligonucleotide probes were diluted at 0.5 μM in 5×SSC, Tween 0.1% andapplied on the glass surface for 2 hours at room temperature. Glassslides were rinsed on a shaker at 37° C., once in 5×SSC for 5 min, andtwice in 2×SSC containing 0.1% SDS for 5 minutes.

Hybridization with radiolabeled probes—Radiolabeled oligonucleotidescomplementary to covalently linked oligonucleotides were used ashybridization probes in order to quantify hybridization yields.Oligonucleotides were enzymatically labeled at their 5′ end terminuswith [γ-³²P)dATP (Amersham, UK) using the bacteriophage T4polynucleotide kinase (New England Biolabs, Beverly, Mass.). Excess(γ-³²P]dATP was removed with a Chroma Spin column TE-10 (Clontech, PaloAlto Calif.). Radiolabeled oligonucleotides (0.5 μM in 5×SSC, Tween0.1%) were then applied onto derivatized slides for 2 hours at roomtemperature. Glass slides were rinsed on a shaker once in 5×SSC for 5min and twice in 2×SSC, SDS 0.1% for 5 minutes at 37° C. Afterhybridization the specific activity was determined by scintillationcounting.

Microscope observation—Glass slides were overlaid with 5×SSC solutionand a micro cover glass. Fluorescence was detected using an invertedmicroscope model Axiovert S100TV, with an arc mercury lamp HBO 100 W/2(Carl Zeiss, Oberkochen, Germany) coupled to a CCD camera equipped witha CCD array Kodak with a format 768(H)×512(V) pixels; 6.91×4.6 mmoverall, pixel size 9×9 μm2 (Princeton Instruments Inc. Trenton, N.J.,USA). Exposition times were between 1 and 50 seconds using the objectiveLD Achroplan 20×/0.400 (Carl Zeiss, Oberkochen, Germany) and filter setsXF22 (Ex: 485DF22, Dichroic: 505DRLPO2 Em: 530DF30) and XF47 (Ex:640DF20, Dichroic: 670DRLPO2 Em: 682DF22) from Omega Optical(Brattleboro Vt.) for FITC and Cy5 fluorophores respectively. Data wereanalyzed using Winwiew software (Princeton Instruments Inc., TrentonN.J., USA).

Results

Evaluation of Storage Stability Attachment and Thermal Stability

We evaluated the storage stability of glass plates prepared with s-MBSand s-SIAB. Since these reagents are sensitive towards hydrolysis,oligonucleotide attachment yields will be dependent on their stability.Amino-derivatized glass plates were functionalized with freshly preparedcrosslinking reagents, s-MBS and s-SIAB. The functionalized slides werestored after cross-linking attachment for 10 days in a dessicator undervacuum in the dark at room temperature. After this time, stored slides(t=10 days) and freshly reacted slides with the cross-linker reagents(t=0) were assayed. The results obtained after reaction of athiol-oligonucleotide and hybridization of a complementary fluorescentprobe were compared for both chemistries at t=0 and time=10 days.

Once immobilized, the s-SIAB-functionalized slides are fully stableafter 10 days storage as evidenced by the same yields of hybridizationobtained at t=0 and t=10 days. In contrast, coupled s-MBS to glass wasfound to be less stable with a 30% loss in yield of oligonucleotideattachment and hybridization after 10 days storage. In conclusion,s-SIAB functionalized slides are preferred as they can be prepared inadvance and stored dry under vacuum in the dark for at least ten dayswithout any reduction in probe attachment yield.

To evaluate the thermal stability of oligonucleotides attached to glass,the slides were subjected to two 5-min treatments at 100° C. in 10 mMTris-HCl, pH 8. The remaining oligonucleotide still immobilized afterwashes was assayed by hybridization with a fluorescently labeledcomplementary oligonucleotide. About 14% of the molecules attached arereleased for s-SIAB glass slides and 17% for S-MBS glass slides afterthe first 5 minutes wash, but no further release was detected in thesecond wash for both chemistries (TABLE 1A). These results areencouraging compared to those obtained by Chrisey et al. 1996, where arelease of more than 62% of oligonucleotides attached on fused silicaslides via the crosslinker SMPB (Succinimidyl4-[p-maleimidophenyl]butyrate) was measured after a 10 min treatment inPBS at 80° C.

TABLE 1A Table 1A: Thermal stability study Hybridisation results(arbitrary units, normalised to 100%) Freshly After 5 min After 2x5 minattached wash at 100° C. wash at 100° C. s-MBS  80 ± 6 69 ± 4 73 ± 4s-SIAB 100 ± 9 84 ± 8 87 ± 3

Oligonucleotides were attached to glass slides functionalized witheither s-MBS or s-SIAB. Attached oligonucleotides were assayed byhybridization with a fluorescently-labeled complementaryoligonucleotide. Fluorescence signal is normalized at 100 for thehighest signal obtained. Averaged values of triplicate analyses arereported.

Hybridization as a Function of Probe Attachment

We have studied the extent of hybridization of covalently boundoligonucleotide probes as a function of the surface coverage of attachedoligonucleotides using the s-MBS, s-SIAB cross-linkers and EDC-mediatedreactions. The concentration of oligonucleotides applied forimmobilization was 1 μM for EDC and has been varied between 1 and 800 μMfor crosslinkers, the surface density was assayed by hybridization with³²P-labeled probes. The optimal concentration for primer attachmentusing the heterobifunctional cross-linkers was 500 μM which equates witha surface density of hybridized molecules of 60 fmol/mm² for s-MBS and270 fmol/mm² for s-SIAB. Similar coverage density as s-MBS was obtainedusing EDC/Imidazole-mediated attachment of 5′-phosphate oligonucleotidesto aminosilanised glass. However, only 1 μM solutions of oligonucleotidewere necessary to attain the same attachment yield, this represents a500-fold excess of oligonucleotide to be attached for the s-MBSchemistry compared to the EDC/imidazole coupling strategy (Table 1B).

TABLE 1B Table 1B: Hybridization as a function of probe attachment Conc.of oligonucleotide Oligo hybridized (fmol/mm²) used for attachment (μM)s-MBS s-SIAB EDC 1 NT NT 50 100 10 100 NT 500 60 270 NT

Oligonucleotides were attached to glass slides functionalized witheither s-MBS or s-SIAB or via mediated activating reagent EDC. Attachedoligonucleotides were assayed by hybridization with a radiolabeledcomplementary oligonucleotide. The specific activity and therefore thedensity of hybridized molecules were determined by scintillation liquid.NT: not tested

The 60 fmol/cm² surface density corresponds to an average molecularspacing between bound oligonucleotides of 8 nm. According to ourresults, a coverage density of 30 fmol/mm² (spacing of 20 nm) issufficient to obtain DNA colonies. This yield can be obtained byimmobilizing primers at 100 μM using the heterobifunctional cross-linkers-SIAB or 1 μM probes using the EDC-mediated approach. The hybridizationdensities we have obtained are in the range of the highest densitiesobtained on glass slides of other grafting protocols previously reported(Guo et al-1994, Joss et al-1997, Beattie et al-1995).

DNA Colony Generation on Glass: Colonies Formation is Dependent on theLength, the Concentration of Template and the Concentration of Primers

Theoretically, DNA colony formation requires an appropriate density ofprimers attached on the surface corresponding to an appropriate lengthof the DNA template. For optimal DNA colony generation, it is importantto define the range of densities of the bound primers and templates, aswell as the stoichiometric ratio between template and primer.

Materials and Methods

Glass Slide Preparation

Glass slides were derivatized and functionalized as described above(Materials and methods). DNA colony primers were CP1 and CP2. The colonytemplates were prepared as described in example 1 for template B′, butusing primers TPB3 and TPB2. The modified colony primers and templateswere applied on glass surface at varying concentrations of both colonyprimer and colony template.

Generation of Colonies

Glass slides stored in 5×SSC were washed in micro-filtered water toremoved salts. Colony growing was initiated on glass surface with a PCRmix; the four dNTP (0.2 mM), 0.1% BSA, 0.1% Tween 20, 1×PCR buffer and0.05 U/μl of AmpliTaq DNA polymerase (Perkin Elmer, Foster City,Calif.). The PCR mix is placed in a frame seal chamber (MJ Research,Watertown, Mass.). The slides were placed in the thermocycler (The DNAEngine, MJ Research Watertown, Mass.) and thermocycling was as carriedout as follows: step 1 at 94° C. for 1 min, step 2 at 65° C. for 3minutes, step 3 at 74° C. for 6 min and this program is repeated 50times. After completion of this program the slides are kept at 6° C.until further use.

Digestion of Double Strand DNA Colonies

The glass surface containing the DNA was cut with a restriction nucleaseby overlaying with the restriction enzyme in a digestion 1× buffer. Thereaction was run twice for 1 h30 at 37° C. Double strand DNA colonieswere denatured by immersing slides 2 times in tris buffer (10 mM, pH 8)at 100° C. for 5 min, followed by a rinse in 5×SSC at 4° C. Slides werestored in 5×SSC for further use.

Hybridization of One Strand DNA Colonies

To prevent non-specific hybridization, glass slides were incubated witha blocking solution (5×SSC, 0.1% Tween, 0.1% BSA) for 1 hour and theslides rinsed in 5×SSC (2 times, 5 min). Fluorescently Cy5 5′ endlabeled oligonucleotide (Eurogentec, Brussels) were diluted at 0.5 μM inSSC 5×, Tween 0.1% and applied to the glass surface for at least 2hours. Glass slides are rinsed on a shaker once in SSC 5× for 5 min andtwice in SSC 5×, SDS 0.1% 5 minutes at 37° C.

The glass slides were visualized as previously described.

We have previously observed that the extent of hybridization is afunction of the density of oligonucleotide attachment. A similar studywith bound DNA templates has shown that colony formation is also afunction of the concentration of template attached on glass slide.Depending on the chemistry used for oligonucleotide and templateattachment, the optimal concentration of template is 1 μM for thebi-functional crosslinkers, s-MBS (FIG. 6B), and 1 nM for EDCcarbodiimide (FIG. 6A). Interestingly, a higher concentration oftemplate does not enhance number of colonies for EDC chemistry and aplateau corresponding to a maximal number of colonies seems to bereached.

We have studied colony formation (number) as a function of theconcentration of primers, concentration of the DNA template applied onthe surface and the length of the DNA template.

We have also evaluated the number of copy of template in each colony.The quantification was based on fluorescence detection with Cy5-, Cy3-or fluorescein-labeled fluorophores supplemented with an anti-bleachingreagent (Prolong, Molecular Probes, Portland Oreg.). The calibration hasbeen done by hybridization experiments on primers attached to thesurface as the exact density corresponding has been determined byradioactivity

Example 5 Colony In-Situ DNA Sequencing

Glass slides (5 mm diameter Verrerie de Carouge, Switzerland) were firstplaced into a Helmanex 0.2% (in H₂O), NaOH 1N bath for 1 h at roomtemperature, rinsed with distilled water, rinsed in pure Hexane, rinsedagain two times with distilled water and treated with HCl 1M over nightat room temperature. Then, they were rinsed two times in distilledwater, and treated with H₂SO₄ (50%)+K₂S₂O₈ for 1 h at room temperature.They were rinsed in distilled water, then two times in Ethanol. Glassslides were derivatized with ATS (as described in example 4).

Colony primers CP1 (5′-pTTTTTTTTTTCACCAACCCAAACCAACCCAAACC) and CP2(5′-pTTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG) which are 5′ phosphorylated(Microsynth GmbH, Switzerland) and DNA template B (prepared as describedin example 1) were 5′ covalently attached onto 5 mm diameter glassslides (Verrerie de Carouge, Switzerland) to a final concentrations of 1μM and 10 nM respectively, as follows: 2 nmoles of each primer wereadded to 0.2 nmoles of template in 1 ml of solution A (41 μl ofMethylimidazole (Sigma, #M-8878) in 50 ml H₂O, pH adjusted to 7 withHCl) and then mixed 1:1 with solution D (0.2 mM EDC in 10 ml of solutionA). On both glass slides sides, 3.5 μl of the mixture were loaded, andincubated over night at room temperature. The glass slides were thenbriefly rinsed with 5×SSC buffer and placed at 100° C. in 10 mM Trisbuffer pH 8.0 for 2×5′.

Non specific sites on glass were blocked with Bovine Serum Albumin (BSA,Boehringer Mannheim GmbH, Germany, #238040) at 1 mg/ml in 5×SSC bufferfor 1 h at room temperature and then rinsed with distilled water.

Glass slides were then individually placed onto a Microamp™ reactiontube (Perkin Elmer) containing 170 μl of PCR mix, and DNA colonies werethen generated using Taq polymerase (AmpliTaq, PE-Applied BiosystemsInc., Foster City Calif.) with 50 cycles (94C/60″, 60C/3′, 72C/6′) in aMTC 200 thermo-cycler (MJ Research, Watertown, Mass.). Each slide wasdigested twice using 1.3 units of Pvu II (Stratagene) in NEB 2 buffer(New England Biolabs) for 45 minutes at 37° C. After digestion, thetubes were placed at 100° C. in 10 mM Tris buffer pH 8.0 for 2×5′, thenblocked with filtered (Millex GV4, Millipore) 1 mg/ml BSA in 2×SSCbuffer for 30′ at room temperature and rinsed first in 2×SSC 0.1% SDSbuffer then in 5×SSC buffer. Each slide was incubated over night at roomtemperature with a 5×SSC/0.1% Tween 20 buffer containing 1 μM of thesequencing primer p181 (CGACAGCCGGAAGGAAGAGGGAGC) overnight at roomtemperature. Controls without primer were kept in 5×SSC 0.1% Tween 20buffer. Glass slides were washed 2 times in 5×SSC 0.1% SDS at 37C for 5′and rinsed in 5×SSC. Primer p181 can hybridize to template B′ and thesequence following p181 is CAGCT . . . . In order to facilitatefocusing, green fluorescent beads have been adsorbed to the bottom ofthe well by incubating each well with 20 μl of a 1/2000 dilution of 200nm yellow/green fluorescent, streptavidin coated FluoSpheres® (MolecularProbes, Eugene, Oreg.) in 5×SSC for 20″ at room temperature.

After hybridization with the primer, 2 μl of a solution containing 0.1%BSA, 6 mM dithiotreitol (Sigma Chemicals), 5 μM Cy5™-dCTP or 5 μMCy5™-dUTP (Amersham, UK) and 1× Sequenase reaction buffer is added toeach slide. The addition of the Cy5™-nucleotide is initiated with theaddition of 1.3 unit of T7 Sequenase™ DNA polymerase (Amersham, UK) fortwo minutes at room temperature. The wells are washed 2 times in5×SSC/0.1% SDS bath for 15′ and rinsed with 5×SSC buffer.

The samples are observed using an inverted microscope (Axiovert S100TV,Carl Zeiss AG, Oberkochen, Germany) equipped with a Micromax 512×768 CCDcamera and Winview software (Princeton Instruments, Trenton, N.J.). Forfocusing, a 20× objective and a XF 22 filter set (Omega Optical,Brattleboro, Vt.) were used, and for observing Cy5™ incorporation on thesamples, a 20× objective and a XF47 filter set (Omega Optical) with a 50second exposure using a 2×2 pixel binning. The yellow/green FluoSpheres®(approximately 100/field of view) do not give a detectable signal usingthe XF47 filter set and 50 second exposure (data not shown). The photosare generated by the program, Winview (Princeton Instruments).

FIG. 7A shows the result after incubation with Cy5™-dCTP on a samplethat has not been incubated with primer p181. One will appreciate, 5blurry spots can be observed, indicating that no dramatic spuriouseffect is taking place (such as Cy5™-dCTP aggregate precipitation,adsorption or simply non specific incorporation to the DNA in thecolonies or on the surface). FIG. 7B shows the result after incubationwith Cy5™-dUTP on a sample that has been incubated with primer p181. Onewill appreciate that no fluorescent spot can be observed, indicatingthat the incorporation of a fluorescent base cannot take place indetectable amounts when the nucleotide proposed for incorporation doesnot correspond to the sequence of the template following the hybridizedprimer. FIG. 7C shows the result after incubation with Cy5™-dCTP on asample that has been incubated with primer p181. One will appreciatethat many fluorescent spots can be observed, indicating that theincorporation of a fluorescent base can indeed take place in detectableamounts when the nucleotide proposed for incorporation does correspondto the sequence of the template following the hybridized primer. Tosummarize, we showed that it is possible to incorporate on a sequencespecific manner fluorescent nucleotides into the DNA contained in thecolonies and to monitor this incorporation with the apparatus and methoddescribed. However, this is only a example. One will appreciate that ifdesired the incorporation of a fluorescent base could be repeatedseveral times. As this is done on a sequence specific manner, it is thuspossible to deduce part of the sequence of the DNA contained in thecolonies.

Example 6 5′ mRNA Sequence Tag Analysis

The most accurate way to monitor gene expression in cells or tissues isto reduce the number of steps between the collection of the sample andthe scoring of the mRNA. New methods for rapidly isolating mRNA arecommercially available. The most efficient methods involve the rapidisolation of the sample and immediate disruption of cells into asolution of guanidinium hydrochloride, which completely disruptsproteins and inactivates RNAses. This is followed by the purification ofthe mRNA from the supernatant of the disrupted cells by oligo-dTaffinity chromatography. Finally, 5′-capped mRNA can be specificallytargeted and transformed into cDNA using a simple strategy (SMART cDNAsynthesis, Clontech, Palo Alto).

This method allows the synthesis of cDNA copies of only thetranslationally active, 5′-capped mRNA. By combining the above rapidmethods of mRNA isolation and cDNA preparation with the grafted-templatemethod of DNA colony generation described in the present application, wehave an approach for the high-throughput identification of a largenumber of 5′ mRNA sequence tags. The advantage of our invention is thepossibility to sequence a large number of cDNA by directly grafting theproduct of the cDNA synthesis reaction, amplifying the cDNA intothousands of copies, followed by the simultaneous in situ sequencing ofthe cDNAs.

Materials and Methods:

Synthetic oligonucleotides and plasmids—Oligonucleotides weresynthesized with 5′-phosphates by Eurogentec or Microsynth. Plasmidscontaining partial coding and 3′-untranslated sequences of the murinepotassium channel gene, mSlo, following the T3 RNA polymerase promoterwere generated by standard methods.

mRNA synthesis—mSlo plasmids were linearized at a single SalI or SacIrestriction nuclease site following the poly A+ sequence in the plasmid.After treatment of the cut plasmid with proteinase K, linear plasmid DNAwas extracted once with phenol/CH₃Cl/isoamyl alcohol and precipitatedwith ethanol. The DNA precipitate was re-dissolved in H₂O at aconcentration of 10 μg/μl. Synthetic mRNA capped with the5′-methylguanosine were synthesized by the mMessage mMachine in vitromRNA synthesis kit as per manufacturer instructions (Ambion, AustinTex.). Synthetic mRNA was stored at 80° C.

Enzymes—Restriction enzymes were obtained from New England Biolabs(Beverly, Mass.).

cDNA synthesis—Synthetic mRNA was mixed with mouse liver poly A+ mRNA atdifferent molar ratios (1:1, 1:10, 1:100) and cDNA synthesis on themixture of synthetic and mouse liver mRNA was performed using the “SMARTPCR cDNA synthesis kit” (Clontech, Palo Alto Calif.) with some minormodifications. In a cDNA reaction, approximately 1 μg of the mRNAmixture was mixed with the -primer CP5, having at the 5′-end thesequence of CPβ, (5′p-AGAAGGAGAAGGAAAGGGAAAGGGTTTTTTTTTTTTTTTTNN). Thisprimer has been used to make the 1st strand cDNA synthesis. For the 2ndstrand synthesis, the “SMART” technique has been used. The basis of theSMART synthesis is the property of the Moloney murine viral reversetranscriptase to add three to five deoxycytosine residues at the3′-termini of first strand cDNA, when the mRNA contains a5′-methylguanosine-cap (SMART user manual, Clontech, Palo Alto Calif.).A CP6 primer, which contains the sequence of CPβ plus AAAGGGGG at the 3′end, (5′p-AGAAGGAGAAGGAAAGGGAAAGGGGG) has been used for the 2nd strandcDNA synthesis. Buffer and SUPERSCRIPT™ II RNase H-reverse transcriptasefrom Moloney murine leukemia virus (Life Technologies, Ltd.) were usedas described in the instructions and the reaction was carried out at 42°C. for 1 hr. The cDNA was assayed by PCR using the primer p251, whichcontains a fragment of the CPβ sequence, (5′-GAGAAGGAAAGGGAAAGG) withTaq DNA polymerase (Platinum Taq, Life Technologies, Ltd.).

Preparation of DNA colonies—The 5′ p-cDNA was mixed with differentconcentrations of the solid phase colony primer, CP2 (5′p-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG) and chemically bound to NucleolinkPCR tubes (NUNC) following manufacturer instructions. DNA colonies werethen generated using Taq polymerase (AmpliTaq Gold, PE-AppliedBiosystems Inc., Foster City Calif.) with 30 cycles (94C/30″, 65C/1′,72C/1.5′) in a MTC 200 thermo-cycler (MJ Research, Watertown, Mass.).

DNA probes and hybridization—³²Biotinylated and ³²P-radiolabelled DNAprobes specific for the mSlo DNA sequence were synthesized with a5′-biotinylated primer and a normal downstream primer by PCR on thetemplate (mSlo plasmid DNA). The probe incorporated α[³²P]-dCTP(Amersham, Amersham UK) at a ratio of 300:1 (α[³²P]-dCTP to dCTP) in thePCR reaction, with a final concentration of the four deoxynucleosidetriphosphates of 50 μM. The resulting biotinylated and radiolabelled DNAprobe was desalted over a Chromaspin-1000 column (Clontech, Palo AltoCalif.). The DNA probes were hybridized to the samples in “easyhyb”buffer (Boehringer-Mannheim, Germany), using the following temperaturescheme (in the MTC200 thermocycler): 94° C. for 5 minutes, followed by68 steps of 0.5° C. decrease in temperature every 30 seconds (in otherwords, the temperature is decreased down to 60° C. in 34 minutes), usingsealed wells. The samples are then washed 3 times with 200 μl of TNT atroom temperature. The wells are then incubated for 30 minutes with 50 μlTNT containing 0.1 mg/ml BSA (New England Biolabs, Beverly, Mass.). Thenthe wells are incubated 5 minutes with 15 μl of solution of redfluorescent, steptavidin-coated, 40 nanometer microspheres (MolecularProbes, Portland, Oreg.). The solution of microspheres is made of 2 μlof the stock solution of microspheres, which have been sonicated for 5minutes in a 50 W ultra-sound water-bath (Elgasonic, Bienne,Switzerland), diluted in 1 ml of TNT solution containing 0.1 mg/ml BSAand filtered with Millex GV4 0.22 μm pore size filter (Millipore,Bedford, Mass.). DNA colony visualization—The stained samples areobserved using an inverted Axiovert 10 microscope using a 20× objective(Carl Zeiss AG, Oberkochen, Germany) equipped with a Micromax 512×768CCD camera (Princeton instruments, Trenton, N.J.), using aPB546/FT580/LP590 filter set, and 10 seconds of light collection. Thefiles are converted to TIFF format and processed in the suitablesoftware (PhotoPaint, Corel Corp. Ltd, Dublin, Ireland). The processingconsisted in inversion and linear contrast enhancement, in order toprovide a picture suitable for black and white printout on a laserprinter.

Results

Synthetic mRNA and cDNA Synthesis

Following cDNA synthesis, the cDNA was checked in a PCR using the p251primer (generated at each end of the first strand cDNA) for the correctlengths of products as assayed by agarose gel electrophoresis. Thesynthetic mSlo mRNA was diluted into the liver mRNA, which was evidencedby the decreasing intensity of the mSlo-specific band and the increaseof a non-specific smear of liver cDNA.

Detection and Quantification of DNA Colonies

DNA colonies were assayed using fluorescent imaging CCD microscopy orscintillation counting. The numbers of fluorescently detectable coloniesincreased as a function of the amount of grafted template, as shown inFIG. 6. This increase was mirrored by the amount of ³²P-radiolabeldetected.

With radiolabelled probes it is possible to detect mRNA copies at about1:100. But with fluorescent microscopic CCD imaging technology, one candetect mRNA to a dilution of 1:10000.

Example 7 Covalent Binding of Primer to the Solid Support (Plastic)

Oligonucleotide primers were attached onto Nucleolink plastic microtitrewells (Nunc, Denmark) in order to determine optimal coupling times andchemistries. Oligonucleotides; CP2(5′-(phosphate)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG),CP8(5′-(amino-hexamethylene)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG), CP9(5′(hydroxyl)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG), CP10(5′-(dimethoxytrityl)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG) and CP11(5′(biotin)-TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG), (Microsynth GmbH,Switzerland), were attached to Nucleolink microtitre wells as follows (8wells each); to each well 20 μl of a solution containing 0.1 μMoligonucleotide, 10 mM 1-methyl-imidazole (pH 7.0) (Sigma Chemicals) and10 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (pH 7.0) (SigmaChemicals) in 10 mM 1-methyl-imidazole. The wells were then sealed andincubated 50° C. for varying amounts of time. The coupling reaction wasterminated at specific times by rinsing twice with 200 μl of RS (0.4 NNaOH, 0.25% Tween 20) and twice with 200 μl TNT (100 mM TrisHCl pH 7.5,150 mM NaCl, 0.1% Tween 20). Tubes were dried at 50° C. for 30′ and werestored in a sealed plastic bag at 4° C.

Stability of Bound Oligonucleotides Under PCR Colony GenerationConditions

Stability was tested under colony growing conditions by adding a PCR mix(20 μl of four dNTPs (0.2 mM), 0.1% BSA, 0.1% Tween 20, 8% DMSO(dimethylsulfoxide, Fluka, Switzerland), 1×PCR buffer). The wells werethen placed in the thermocycler and for 33 repetitions under thefollowing conditions: 94° C. for 45 seconds, 60° C. for 4 minutes, 72°C. for 4 minutes. After completion of this program, the wells wererinsed with 5×SSC, 0.1% Tween 20 and kept at 8° C. until further use.Prior to hybridization wells are filled with 50 μl 5×SSC, 0.1% Tween 20heated at 94° C. for 5 minutes and stored at RT.

Probe:

Oligonucleotide probes, R57 (5′(phosphate)-GTTTGGGTTGGTTTGGGTTGGTG,control probe) and R58 (5′-(phosphate)-CCCTTTCCCTTTCCTTCTCCTTCT, whichis complementary to CP2, CP8, CP9, CP10 and CP11) were enzymaticallylabeled at their 5′ end terminus with [γ-³²P]dATP (Amersham, UK) usingthe bacteriophage T4 polynucleotide kinase (New England Biolabs,Beverly, Mass.). Excess ³²P dATP was removed with a Chroma Spin columnTE-10 (Clontech, Palo Alto Calif.). Radiolabeled oligonucleotides (0.5μM in 5×SSC, 0.1% Tween 20) were then hybridized to the oligonucleotidederivatized Nucleolink wells at 37° C. for two hours. The wells werewashed 4 times with 5×SSC, 0.1% Tween 20 at room temperature, followedby a wash with 0.5×SSC, 0.1% Tween 20 for 15′ at 37° C. Wells were thenassayed for bound probe by scintillation counting.

Results

There is a marked difference in the rate and specificity ofoligonucleotide coupling depending on the nature of 5′-functional groupon the oligonucleotide. Oligonucleotides carrying the 5′-amino groupcoupled approximately twice as fast as oligonucleotides functionalizedwith a 5′-phosphate group (see Table 2 and FIG. 8). In addition, thecontrol oligonucleotides functionalized with 5′ hydroxyl, 5′-DMT or5′-biotin all coupled at rates similar to that of the 5′-phosphate,which questions the 5′ specific nature of the chemical attachment usingthe 5′-phosphate group.

TABLE 2 5′- 5′- 5′- 5′- 5′- phosphate amino hydroxyl DMT biotin Ka(min−1) 0.0068 0.0135 0.0063 0.0070 0.0068 Attached 608 1344 542 602 650oligo- nucleotide (fmol/well) PCR stability 56 69 66 66 62 (% remaining)

The invention claimed is:
 1. A method for parallel amplification of acollection of single nucleic acid molecules having different sequencescomprising: a) providing a vessel comprising the collection of singlenucleic acid molecules, b) treating the single nucleic acid molecules inthe vessel by attaching a known, common sequence at the 5′ and 3′ endsof each nucleic acid molecule of the collection, thereby generating acollection of treated nucleic acid molecules wherein each of thecollection of treated nucleic acid molecules contains a differentsequence and the known, common sequence at its 5′ and 3′ ends, c)separating the collection of treated nucleic molecules such thatindividual molecules from the collection of treated nucleic acidmolecules are separated from each other, and d) performing a singleamplification reaction in the presence of forward colony primers andreverse colony primers complementary to and/or identical to the knowncommon sequence, wherein at least one of the colony primers isimmobilized on a solid support, thereby simultaneously amplifying thedifferent sequence of each of the treated nucleic acid moleculesseparated in step c) under conditions such that a plurality of nucleicacid colonies is formed on the solid support and the collection ofsingle nucleic acid molecules having different sequences is amplified.2. The method according to claim 1, wherein said forward colony primersand said reverse colony primers are immobilised on a solid support. 3.The method according to claim 1, wherein said solid support is a bead ormicroparticle.
 4. The method according to claim 1, wherein said solidsupport is a planar surface.
 5. A method for parallel amplification of acollection of single nucleic acid molecules having different sequencescomprising: a) treating the collection of nucleic acid molecules therebygenerating a collection of treated nucleic acid molecules wherein eachof the collection of treated nucleic acid molecules contains a differentsequence and a known, common sequence at its 5′ and 3′ ends, b)immobilizing the treated nucleic acid molecules on a solid support,thereby separating the collection of treated nucleic acid molecules suchthat individual treated nucleic acid molecules from the collection oftreated nucleic acid molecules are separated from each other, and c)performing an amplification reaction in the presence of forward colonyprimers and reverse colony primers complementary to and/or identical tothe known common sequence, wherein at least one of the colony primers isimmobilized directly to the solid support, thereby simultaneouslyamplifying the different sequence of each of the treated nucleic acidmolecules separated in step b) under conditions such that a plurality ofnucleic acid colonies is formed on the solid support and the collectionof single nucleic acid molecules having different sequences isamplified.
 6. The method according to claim 5, wherein said forwardcolony primers and said reverse colony primers are immobilised on asolid support.
 7. The method according to claim 5, wherein said solidsupport is a bead or microparticle.
 8. The method according to claim 5,wherein said solid support is a planar surface.
 9. A method for parallelamplification of a collection of single nucleic acid molecules havingdifferent sequences comprising: a) treating the collection of nucleicacid molecules thereby generating a collection of treated nucleic acidmolecules wherein each of the collection of treated nucleic acidmolecules contains a different sequence and a known, common sequence atits 5′ and 3′ ends, b) immobilizing the treated nucleic acid moleculeson a solid support, the solid support comprising a lawn of immobilizedcolony primers comprising forward colony primers and/or reverse colonyprimers, the colony primers being complementary to and/or identical tothe known common sequence, thereby separating the collection of treatednucleic acid molecules such that individual treated nucleic acidmolecules from the collection of treated nucleic acid molecules areimmobilized at intervals within the lawn of immobilized colony primers,and c) performing an amplification reaction in the presence of theforward colony primers and/or the reverse colony primers therebysimultaneously amplifying the different sequence of each of the treatednucleic acid molecules separated in step b) under conditions such that aplurality of nucleic acid colonies is formed on the solid support andthe collection of single nucleic acid molecules having differentsequences is amplified.
 10. The method of claim 9, wherein the lawn ofimmobilized colony primers comprises forward colony primers and reversecolony primers.
 11. The method of claim 9, further comprising anadditional step of performing at least one step of sequencedetermination of nucleic acids in one or more of the nucleic acidcolonies.
 12. The method of claim 9, wherein the density of the nucleicacid colonies on the solid support is 10,000/mm² to 100,000/mm².